Kun-Han Chuang1, Fengshan Liang1, Ryan Higgins1, Yanchang Wang2. 1. Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306-4300. 2. Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306-4300 yanchang.wang@med.fsu.edu.
Abstract
Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.
Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by humanubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.
Protein misfolding occurs spontaneously under normal physiological conditions, but conditions such as genetic mutations, environmental insults, and oxidative stress also stimulate it. Misfolded proteins are cytotoxic, but cells have developed protein quality control systems to restore correct protein folding or degrade misfolded proteins. The chaperone network prevents aggregation of misfolded proteins and assists correct refolding (Hartl ). If refolding fails, misfolded proteins can be modified by ubiquitination, which leads to their degradation by the ubiquitin proteasome system (Varshavsky, 2012). Moreover, misfolded proteins can form inclusion bodies (IBs) that are cleared by lysosomes (Kroemer ). For example, in patients with Huntington’s disease (HD), neuron cells have IBs that contain misfolded huntingtin (Chiti and Dobson, 2006). Increasing evidence suggests that compromised cellular capacity to dispose misfolded proteins directly contributes to the onset of numerous neurodegenerative diseases.HD is a neurodegenerative disease with symptoms such as frontal cognitive deficits and involuntary abnormal movements (Walker, 2007). This disease is attributed to the expansion of CAG repeat (encoding glutamine) within the exon 1 of the huntingtin (HTT) gene, which causes terminal misfolding of the Htt protein. The HTT gene in normal individuals has 6–35 CAG repeats (polyQ), but expansions of >40 CAG repeats in the HTT gene cause HD (Andrew ). The sequestration of misfolded proteins into IBs is believed to alleviate their cytotoxicity and facilitate their disposal by lysosomes (Chin ; Tyedmers ).The huntingtin gene locus in the human genome spans 180 kb and consists of 67 exons. The transgenic R6/2 mouse is the most commonly used animal model of HD and expresses N-terminally truncated mutant humanHtt with 125 polyQ repeats within exon 1. R6/2 mice develop HD-like symptoms, including motor and cognitive deficits (Mangiarini ). Expression of truncated Htt with polyQ expansion and a proline-rich domain is sufficient to induce protein aggregation in vitro (Muchowski ). Of interest, expression of this fragment in yeast and mammalian cells also results in IB (aggresome) formation, and this expression is well tolerated. However, expression of this Htt fragment lacking the proline-rich domain fails to form IBs and causes cytotoxicity in yeast cells, supporting the notion that IB formation alleviates the cytotoxicity of misfolded proteins (Krobitsch and Lindquist, 2000; Meriin ; Wang ). The capability of IB formation makes yeast an ideal model organism in which to study the biological processes of IB formation because of the availability of numerous genetic tools for yeast.Previous studies indicate the ubiquitination of mutated Htt proteins. Striatum from HD brains showed elevated levels of ubiquitinated Htt N-terminal fragments (Mende-Mueller ). The N-terminal domain of Htt with 44 polyQ repeats interacts with ubiquitination enzymes, which may contribute to Htt ubiquitination (Kalchman ). The three lysine residues in the N-terminus of Htt are responsible for this modification, but the same residues are also subject to SUMOylation (Steffan ). SUMOylation stabilizes Htt and reduces its ability to form aggregates, presumably by competing for ubiquitination. Recent evidence indicates that K48-linked ubiquitination of the N-terminal Htt fragments leads to proteasome-mediated degradation. However, in HD knock-in mice, aging decreases K48-linked ubiquitination and proteasome-mediated degradation of mutated Htt, whereas it increases K63-linked ubiquitination, indicating the complexity of the ubiquitination of misfolded proteins (Bhat ).Ubiquitinated proteins can be degraded through direct binding to proteasome receptors such as Rpn10 and Rpn13 (Husnjak ). Ubiquitinated proteins can also interact with ubiquitin-like domain (UBL)–ubiquitin-associated (UBA) receptors, which subsequently transport the client proteins to proteasomes for degradation. UBL-UBA proteins contain an N-terminal UBL and either one or two UBA domains at the C-terminal (Kang ). Previous work indicates that the human UBL-UBA proteins ubiquilin 1 and 2 are associated with various pathological inclusions, and mutations in ubiquilin 2 cause inherited amyotrophic lateral sclerosis (Daoud and Rouleau, 2011; Deng ). Other studies suggest that ubiquilin 2 preferentially associates with Htt-polyQ aggregates compared with other protein inclusions (Doi ; Wang and Monteiro, 2007; Rutherford ). Some work indicated that ubiquilin proteins transport ubiquitinated substrates to the proteasome for degradation (Wang and Monteiro, 2007), and other research suggested that ubiquilin proteins facilitate IB formation, which may promote lysosome-medicated protein clearance (Heir ). However, the molecular function of ubiquilin proteins in proteasome- or lysosome-mediated clearance of misfolded proteins remains largely unknown.Here we used Htt exon 1 with 103-polyQ expansion and the proline-rich region (Htt103QP) as a model substrate to study the function of yeast ubiquilin, Dsk2, in the clearance of misfolded proteins. We found that dsk2Δ mutants exhibited slow growth and failed to form IB efficiently when Htt103QP was overexpressed. Our results indicate that the UBL domain of Dsk2 provides the functional specificity in Htt103QP IB formation. Surprisingly, Dsk2 is dispensable for proteasome-mediated protein degradation but is required for efficient delivery of Htt103QP into vacuoles (lysosomes), indicating that ubiquilin/Dsk2 facilitates lysosome-mediated clearance of mutated Htt by promoting IB formation. Furthermore, the rescue of the dsk2Δ mutant phenotype by humanubiquilin 1 and 2 indicates the functional conservation of this protein family.
RESULTS
Dsk2 is required for mutated huntingtin inclusion body formation in budding yeast
IB formation is believed to be cytoprotective because it sequesters toxic misfolded proteins (Wang ; Gong ). In budding yeast, expression of mutated Htt with polyQ expansion leads to IB formation, but failure in this process causes toxicity to yeast cells (Duennwald ; Wang ). Therefore we speculated that yeast mutants with defective IB formation should exhibit slow growth when mutated Htt is expressed. In this context, we constructed a yeast strain harboring mfa1:PMFA1-Sphis5 and an integrated plasmid that contains Htt with 103-polyQ expansion and the proline-rich region (Htt103QP). This Htt fragment was tagged with Flag at the N-terminus and green fluorescent protein (GFP) at the C-terminus and is under control of a galactose promoter (P; hereafter Htt103QP). This strain was crossed with all of the ∼4800 yeast deletion mutants from ATCC (Manassas, VA), and the haploid cells (MATa) containing a gene deletion and Htt103QP were selected (Tong ; Daniel ). The resulting ∼4800 mutants with Htt103QP were examined for their growth on glucose and galactose plates (Figure 1A). We also used a query strain with P as a control to exclude the mutants that fail to grow on galactose plates. IB formation of the selected strains was examined after Htt103QP induction. From this genome-wide screen, we identified some yeast mutants that exhibited slow growth on galactose plates and an IB formation defect, including dsk2Δ mutants.
FIGURE 1:
The absence of Dsk2 results in defects in Htt103QP IB formation. (A) The identification of yeast mutants sensitive to Htt103QP overexpression. About 4800 yeast deletion mutants with P (Htt103QP) were spotted onto glucose or galactose plates for growth assay. After 2 d of incubation at 30°C, the growth was examined. (B) dsk2Δ mutants are sensitive to Htt103QP overexpression. WT and dsk2Δ, rad23Δ, and ddi1Δ mutant cells were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates for 2 d of incubation at 30°C. (C) dsk2Δ mutant cells exhibit defective Htt103QP IB formation. WT and dsk2∆, rad23∆, and ddi1∆ mutant cells with Htt103QP were grown in galactose medium at 30°C for 12 h to induce Htt103QP expression. Top, fluorescence microscopy, the GFP signal in representative cells. Bottom, percentage of cells with different patterns of Htt103QP-GFP signal (n > 100). The result is the average of three independent experiments. Scale bar, 5 μm. (D) dsk2∆ mutant cells show more soluble Htt103QP. Cells were grown in galactose medium at 30°C for 12 h to prepare cell lysates using a bead beater. The cell lysates were centrifuged and divided into detergent-soluble (supernatant) and detergent-insoluble (pellet) fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. The Pgk1 levels are used as a loading control.
The absence of Dsk2 results in defects in Htt103QP IB formation. (A) The identification of yeast mutants sensitive to Htt103QP overexpression. About 4800 yeast deletion mutants with P (Htt103QP) were spotted onto glucose or galactose plates for growth assay. After 2 d of incubation at 30°C, the growth was examined. (B) dsk2Δ mutants are sensitive to Htt103QP overexpression. WT and dsk2Δ, rad23Δ, and ddi1Δ mutant cells were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates for 2 d of incubation at 30°C. (C) dsk2Δ mutant cells exhibit defective Htt103QP IB formation. WT and dsk2∆, rad23∆, and ddi1∆ mutant cells with Htt103QP were grown in galactose medium at 30°C for 12 h to induce Htt103QP expression. Top, fluorescence microscopy, the GFP signal in representative cells. Bottom, percentage of cells with different patterns of Htt103QP-GFP signal (n > 100). The result is the average of three independent experiments. Scale bar, 5 μm. (D) dsk2∆ mutant cells show more soluble Htt103QP. Cells were grown in galactose medium at 30°C for 12 h to prepare cell lysates using a bead beater. The cell lysates were centrifuged and divided into detergent-soluble (supernatant) and detergent-insoluble (pellet) fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. The Pgk1 levels are used as a loading control.Dsk2, Rad23, and Ddi1 are the three UBL-UBA proteins in budding yeast that function as ubiquitin receptors to transport ubiquitinated proteins to the proteasome (Kang ). To our surprise, we did not identify rad23Δ and ddi1Δ mutants from this screen. Therefore we first compared the growth of these three mutants and wild-type (WT) cells containing Htt103QP on glucose and galactose plates. Consistent with our screen, we noticed the slow growth of dsk2Δ mutants on galactose plates, but rad23Δ and ddi1Δ mutants grew similarly to WT cells (Figure 1B), indicating distinct roles of these three UBL-UBA proteins in response to the expression of Htt103QP. Next we examined the GFP signal in dsk2Δ, rad23Δ, and ddi1Δ mutants after induction of Htt103QP-GFP expression in galactose medium at 30°C for 12 h. Most of the WT cells (>80%) showed one bright GFP aggregate with variable sizes. rad23Δ and ddi1Δ mutant cells exhibited a similar phenotype as WT cells, but only 54% of dsk2Δ mutant cells showed a single GFP aggregate. Some dsk2Δ cells showed multiple tiny GFP aggregates or a mixture of tiny and big aggregates. Moreover, enhanced GFP background was observed in most dsk2∆ cells, indicating defective IB formation (Figure 1C).Previous work demonstrated enriched distribution of mutated Htt in the detergent-insoluble fraction, presumably due to IB formation (Taylor ; Gong ). Moreover, the soluble mutated Htt oligomers before IB formation are cytotoxic (Takahashi ; Lajoie and Snapp, 2010). Therefore we examined Htt103QP protein levels in supernatant (soluble fraction) and pellet (insoluble fraction) from WT, dsk2Δ, rad23Δ, and ddi1Δ mutant cells after induction of Htt103QP expression for 12 h. In WT cells, the majority of Htt103QP was detected in the pellet fraction, which is consistent with efficient IB formation. However, much less Htt103QP protein was detected in the pellet fraction from dsk2Δ mutant cells. In clear contrast, rad23Δ and ddi1Δ mutants showed similar distribution of Htt103QP as WT cells (Figure 1D). Taken together, these results indicate that Dsk2 promotes Htt103QP IB formation in budding yeast, and we speculate that the defect in Htt103QP IB formation causes slow growth of dsk2Δ mutants on galactose plates.
The UBL domain of Dsk2 is essential for inclusion body formation
Our results suggest that Dsk2, but not Rad23 and Ddi1, is required for Htt103QP IB formation in yeast cells. Next we examined which domain of Dsk2 contributes to this unique function. Dsk2, Rad23, and Ddi1 contain a UBL and a UBA domain that binds to the proteasome and ubiquitinated proteins, respectively (Verma ). In addition, Dsk2 contains a Sti1-like repeat sequence (STI), which is found in proteins that bind to heat shock chaperones (Chang ; Lassle ). The STI domain in the human ubiquilin mediates the association with misfolded proteins (Stieren ). We deleted the UBL or STI domain from the yeast genome to generate dsk2Δ and dsk2Δ mutants, respectively, and then examined the sensitivity of these mutants to Htt103QP overexpression, as well as IB formation (Figure 2A). Because the UBA domain of Dsk2 has been shown to function as a stabilizing signal to protect Dsk2 from degradation (Heinen ), we did not generate a dsk2 mutant with UBA deletion. Like dsk2Δ, dsk2Δ mutant cells with Htt103QP exhibited similar slow-growth phenotype on galactose plates, but dsk2Δ mutant cells grew similarly to WT cells (Figure 2B). Consistently, dsk2Δ mutant cells exhibited an obvious IB formation defect but one that was a little less dramatic than for dsk2Δ mutants. However, IB formation in dsk2Δ mutant cells was similar to that in WT cells (Figure 2C). We further examined the distribution of Htt103QP in the supernatant and pellet fractions in these dsk2 mutants after Htt103QP induction for 12 h. Most of the Htt103QP was detected in the insoluble pellet fraction in WT cells, but much less Htt103QP was detected in the pellet fraction in dsk2Δ and dsk2Δ mutant cells. In contrast, dsk2Δ mutant cells showed similar distribution of Htt103QP in supernatant and pellet fractions as WT cells, which is consistent with efficient IB formation (Figure 2D). These data indicate that the UBL domain of Dsk2 is critical for its function in IB formation.
FIGURE 2:
Deletion of the DSK2 UBL domain causes defective Htt103QP IB formation. (A) Schematic illustration of the domains of the yeast Dsk2 protein. (B) dsk2∆ and dsk2∆ mutants show sick growth when Htt103QP is overexpressed. Cells were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates for 2 d of incubation at 30°C. (C) dsk2∆ mutants show defective Htt103QP IB formation similar to dsk2∆ mutants. WT and dsk2∆, dsk2Δ, and dskΔ mutant cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in some representative cells by fluorescence microscopy. Bottom, percentage of cells with different patterns of GFP distribution (n > 100). The result is the average of three separate experiments. Scale bar, 5 μm. (D) dsk2Δ and dsk2Δ mutants show more detergent-soluble Htt103QP. Cells with the indicated genotypes were grown in galactose medium for 12 h. Cell lysates were prepared and divided into detergent-soluble (supernatant) and -insoluble (pellet) fractions after centrifugation. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1 levels were used as a loading control.
Deletion of the DSK2 UBL domain causes defective Htt103QP IB formation. (A) Schematic illustration of the domains of the yeastDsk2 protein. (B) dsk2∆ and dsk2∆ mutants show sick growth when Htt103QP is overexpressed. Cells were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates for 2 d of incubation at 30°C. (C) dsk2∆ mutants show defective Htt103QP IB formation similar to dsk2∆ mutants. WT and dsk2∆, dsk2Δ, and dskΔ mutant cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in some representative cells by fluorescence microscopy. Bottom, percentage of cells with different patterns of GFP distribution (n > 100). The result is the average of three separate experiments. Scale bar, 5 μm. (D) dsk2Δ and dsk2Δ mutants show more detergent-soluble Htt103QP. Cells with the indicated genotypes were grown in galactose medium for 12 h. Cell lysates were prepared and divided into detergent-soluble (supernatant) and -insoluble (pellet) fractions after centrifugation. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1 levels were used as a loading control.It is possible that the unique role of Dsk2 in IB formation is due to the specific binding of Htt103QP to Dsk2 but not to Rad23 and Ddi1. Thus we examined the interaction between Htt103QP and these three UBL-UBA proteins. For this purpose, we generated strains expressing Flag-Htt103QP-GFP and Myc-tagged Dsk2, Rad23, or Ddi1. After induction of Htt103QP in galactose medium for 4 h, when a majority of the cells did not form IBs, the cells were collected to prepare cell lysates. The coimmunoprecipitation results using anti-Myc antibody showed that Htt103QP binds to Dsk2, Rad23, and Ddi1 with similar affinity (Figure 3A). Therefore the function of Dsk2 in IB formation is unlikely to be attributed to its specific binding to Htt103QP.
FIGURE 3:
Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 hybrid proteins show normal Htt103QP IB formation. (A) Dsk2, Rad23, and Ddi1 interact with Htt103QP. Cells expressing 13×Myc-tagged Dsk2, Rad23, or Ddi1, as well as Flag-Htt103QP, were grown in galactose medium at 30°C for 4 h. The cell lysates were pulled down with anti-Myc antibody. The protein levels of Myc-tagged Dsk2, Rad23, and Ddi1 and Flag-tagged Htt103QP in the cell lysates and immunoprecipitates were analyzed by Western blotting. (B) Schematic illustration of the domains in the constructed chimeras. To construct yeast strains expressing chimeras UBLDsk2-Rad23 and UBLDsk2-Ddi1, we replaced the C-terminal part of DSK2 gene (except the UBL domain) from the yeast genome with the corresponding sections of RAD23 and DDI1 genes using PCR-based homologous recombination. The resulting strains express the chimeras from the endogenous DSK2 promoter. (C) Cells expressing chimeras UBLDsk2-Rad23 and UBLDsk2-Ddi1 exhibit similar growth as WT cells when Htt103QP is overexpressed. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates, which were incubated at 30°C for 2 d. (D) Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 chimera show similar Htt103QP distribution in soluble and insoluble factions as WT cells. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown in galactose medium at 30°C for 12 h. Cells lysates from these cells were centrifuged and divided into detergent-soluble (supernatant) and -insoluble (pellet) fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1 levels are used as a loading control. (E) Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 chimeras show similar Htt103QP IB formation as WT cells. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in these cells by fluorescence microscopy. Bottom, average percentage of cells with different GFP distribution patterns (n > 100). The results are the average of three separate experiments. Scale bar, 5 μm.
Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 hybrid proteins show normal Htt103QP IB formation. (A) Dsk2, Rad23, and Ddi1 interact with Htt103QP. Cells expressing 13×Myc-tagged Dsk2, Rad23, or Ddi1, as well as Flag-Htt103QP, were grown in galactose medium at 30°C for 4 h. The cell lysates were pulled down with anti-Myc antibody. The protein levels of Myc-tagged Dsk2, Rad23, and Ddi1 and Flag-tagged Htt103QP in the cell lysates and immunoprecipitates were analyzed by Western blotting. (B) Schematic illustration of the domains in the constructed chimeras. To construct yeast strains expressing chimeras UBLDsk2-Rad23 and UBLDsk2-Ddi1, we replaced the C-terminal part of DSK2 gene (except the UBL domain) from the yeast genome with the corresponding sections of RAD23 and DDI1 genes using PCR-based homologous recombination. The resulting strains express the chimeras from the endogenous DSK2 promoter. (C) Cells expressing chimeras UBLDsk2-Rad23 and UBLDsk2-Ddi1 exhibit similar growth as WT cells when Htt103QP is overexpressed. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown to saturation, 10-fold diluted, and spotted onto glucose and galactose plates, which were incubated at 30°C for 2 d. (D) Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 chimera show similar Htt103QP distribution in soluble and insoluble factions as WT cells. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown in galactose medium at 30°C for 12 h. Cells lysates from these cells were centrifuged and divided into detergent-soluble (supernatant) and -insoluble (pellet) fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1 levels are used as a loading control. (E) Cells expressing UBLDsk2-Rad23 or UBLDsk2-Ddi1 chimeras show similar Htt103QP IB formation as WT cells. WT, dsk2∆, UBL, and UBL cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in these cells by fluorescence microscopy. Bottom, average percentage of cells with different GFP distribution patterns (n > 100). The results are the average of three separate experiments. Scale bar, 5 μm.To determine further the role of the Dsk2 UBL domain in IB formation, we generated yeast strains expressing chimeric proteins UBLDsk2-Rad23 and UBLDsk2-Ddi1, in which the C-terminal part, but not the UBL domain of DSK2 gene, was replaced with the corresponding fractions of RAD23 and DDI1 genes (Figure 3B). We directly replaced the C-terminal fraction of DSK2 gene in the yeast genome with the PCR products of the corresponding RAD23 and DDI1 gene fragments that also contain a selectable marker. Therefore the resulting strains express chimeric genes UBL and UBL under the control of the endogenous DSK2 promoter. We first examined the growth on galactose plates of these strains containing Htt103QP. Yeast cells expressing UBLDsk2-Rad23 and UBLDsk2-Ddi1 chimeras grew similarly to WT cells on galactose plates (Figure 3C). Moreover, these cells showed efficient IB formation like WT cells (Figure 3E). Finally, these cells also showed enriched distribution of Htt103QP in the pellet fraction like WT cells (Figure 3D). Therefore our data support the conclusion that the UBL domain of Dsk2 contributes to its functional specificity in promoting Htt103QP IB formation.
Dsk2 is not required for proteasome-dependent degradation of Htt103QP
UBL-UBA proteins transport ubiquitinated proteins to proteasomes for degradation (Lowe ). To test this possibility for Htt103QP, we first examined the protein stability of Htt103QP after a short period of induction, when no IB formation was noticed. We also examined the effect of the proteasome inhibitor MG132 on the stability of Htt103QP. To enhance MG132 permeability of yeast cells, we grew cells in medium containing 0.1% l-proline and added 0.003% SDS into the medium for 3 h before MG132 instillation as described (Liu ). After Htt103QP induction in galactose medium for 1 h, glucose was added to the medium to shut off Htt103QP expression, and the Htt103QP protein level was analyzed over time. After 1 h of induction in galactose medium, the expression level of Htt103QP was high, but no IBs were observed. After addition of glucose in the medium, Htt103QP protein level declined over time, indicating efficient degradation. However, the presence of the proteasome inhibitor MG132 caused dramatic Htt103QP stabilization (Figure 4A). Using the same protocol, we found that MG132 treatment delayed the degradation of S-phase cyclin Clb5 (Figure 4B), indicating that MG132 inhibits proteasome activity under this experimental condition. Therefore Htt103QP is likely subject to proteasome-mediated degradation after a short period of induction.
FIGURE 4:
Htt103QP degradation after a short period of induction (1 h) is independent of Dsk2. (A) The degradation of Htt103QP after a short induction in the absence and presence of proteasome inhibitor. WT cells with Htt103QP plasmid growing in raffinose medium containing 0.1% l-proline at 30°C were pretreated with 0.003% SDS. After 3 h, dimethyl sulfoxide (DMSO) or 75 μM MG132 was added into the cultures. After incubation for 30 min, galactose (2%) was added into the medium to induce Htt103QP expression for 1 h. Finally, glucose (2%) was added into the cultures to shut off Htt103QP expression. The cells were collected over time to examine Htt103QP protein levels by Western blotting. Pgk1 levels were used as a loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off. (B) Cell cycle protein Clb5 is stabilized in the presence of MG132. Yeast cells with Clb5-HA were treated as described. The Clb5 proteins levels were examined at the indicated time points after glucose addition. Right, ratio change. (C) dsk2Δ mutants show similar Htt103QP degradation kinetics as WT cells after a short induction. WT and dsk2Δ, rad23∆, and ddi1∆ mutant cells with Htt103QP plasmid were grown in 30°C raffinose medium to log phase, and then galactose (2%) was added to induce Htt103QP expression. After 1 h of induction, glucose (2%) was added into the medium to shut off Htt103QP expression. Cells were collected at the indicated times, and Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off.
Htt103QP degradation after a short period of induction (1 h) is independent of Dsk2. (A) The degradation of Htt103QP after a short induction in the absence and presence of proteasome inhibitor. WT cells with Htt103QP plasmid growing in raffinose medium containing 0.1% l-proline at 30°C were pretreated with 0.003% SDS. After 3 h, dimethyl sulfoxide (DMSO) or 75 μM MG132 was added into the cultures. After incubation for 30 min, galactose (2%) was added into the medium to induce Htt103QP expression for 1 h. Finally, glucose (2%) was added into the cultures to shut off Htt103QP expression. The cells were collected over time to examine Htt103QP protein levels by Western blotting. Pgk1 levels were used as a loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off. (B) Cell cycle protein Clb5 is stabilized in the presence of MG132. Yeast cells with Clb5-HA were treated as described. The Clb5 proteins levels were examined at the indicated time points after glucose addition. Right, ratio change. (C) dsk2Δ mutants show similar Htt103QP degradation kinetics as WT cells after a short induction. WT and dsk2Δ, rad23∆, and ddi1∆ mutant cells with Htt103QP plasmid were grown in 30°C raffinose medium to log phase, and then galactose (2%) was added to induce Htt103QP expression. After 1 h of induction, glucose (2%) was added into the medium to shut off Htt103QP expression. Cells were collected at the indicated times, and Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off.Next we assessed whether UBL-UBA proteins facilitate Htt103QP degradation after a short induction. For this purpose, we compared the Htt103QP degradation kinetics in WT, dsk2Δ, rad23Δ, and ddi1Δ mutants after 1 h of induction. The Htt103QP levels decreased over time in WT cells after the induction was shut off by glucose (Figure 4C). Of interest, rad23Δ but not dsk2Δ and ddi1Δ mutants showed compromised Htt103QP degradation (Figure 4C). This result suggests that Dsk2 and Ddi1 are dispensable for Htt103QP degradation after a short induction, but that cells need Rad23 for efficient Htt103QP degradation, presumably by transporting Htt103QP to proteasomes.The efficient degradation of Htt103QP proteins before IB formation is likely through the proteasome. IB formation is observed in yeast cells after Htt103QP induction for ∼5 h, and Htt103QP proteins inside IBs might be degraded via distinct mechanisms, such as autophagy-dependent vacuolar degradation. If that is the case, the IB formation defect in dsk2Δ cells will impede Htt103QP clearance through the autophagy pathway. To test this possibility, we first induced Flag-Htt103QP-GFP expression in galactose medium for 12 h, which led to IB formation in WT cells. Then we added glucose and hydroxyurea (HU) into the cultures to shut off Htt103QP expression and block the cell cycle. We examined the GFP signal over time. Before glucose addition, 80% of WT cells showed IB structure, but IB structure was present in only 17% of cells after glucose addition for 8 h, and no obvious GFP signal was observed in other cells. Before glucose addition, dsk2∆ cells exhibited less efficient IB formation (37%), but 38% of cells showed multiple GFP dots. After glucose addition for 8 h, 26% of dsk2∆ cells still showed GFP aggregates (Figure 5A), indicating the less efficient clearance of Htt103QP-GFP in dsk2Δ mutants.
FIGURE 5:
Htt103QP degradation is compromised in dsk2Δ mutants after IB formation. (A) Delayed clearance of Htt103QP-GFP signal in dsk2Δ mutants after a long period of induction. WT and dsk2Δ mutant cells were grown in galactose medium for 12 h at 30°C. After addition of glucose (2%) and HU (200 mM) into the cultures to shut off Htt103QP expression and block cell cycle progression, cells were collected at the indicated time points to examine the GFP signal by fluorescence microscopy. Top, GFP signal in representative cells at 0, 4, and 8 h after glucose addition. Bottom, percentage of cells with different GFP patterns (n > 100). Scale bar, 5 μm. (B) Htt103QP degradation kinetics in WT and dsk2Δ cells after a long induction. WT and dsk2Δ mutant cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C. The cells were pretreated with 0.003% SDS for 3 h, and then DMSO or 75 μM MG132 was added into the cultures. After incubation for 30 min, glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. Cells were collected at the indicated times, and Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control. Right, ratio change of Htt103QP to Pgk1 over time.
Htt103QP degradation is compromised in dsk2Δ mutants after IB formation. (A) Delayed clearance of Htt103QP-GFP signal in dsk2Δ mutants after a long period of induction. WT and dsk2Δ mutant cells were grown in galactose medium for 12 h at 30°C. After addition of glucose (2%) and HU (200 mM) into the cultures to shut off Htt103QP expression and block cell cycle progression, cells were collected at the indicated time points to examine the GFP signal by fluorescence microscopy. Top, GFP signal in representative cells at 0, 4, and 8 h after glucose addition. Bottom, percentage of cells with different GFP patterns (n > 100). Scale bar, 5 μm. (B) Htt103QP degradation kinetics in WT and dsk2Δ cells after a long induction. WT and dsk2Δ mutant cells with Htt103QP plasmid were grown in galactose medium for 12 h at 30°C. The cells were pretreated with 0.003% SDS for 3 h, and then DMSO or 75 μM MG132 was added into the cultures. After incubation for 30 min, glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. Cells were collected at the indicated times, and Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control. Right, ratio change of Htt103QP to Pgk1 over time.We further compared Htt103QP protein level in WT and dsk2Δ mutants. After cells were grown in galactose for 12 h, the cells were pretreated with 0.003% SDS, followed by the addition of proteasome inhibitor MG132 into some cultures for 30 min. Glucose and HU were then added into the medium, and cells were collected to determine Htt103QP levels. In WT cells, Htt103QP protein levels decreased over time, and MG132 treatment delayed but did not block Htt103QP clearance (Figure 5B), indicating that functional proteasomes may contribute partially to the clearance. dsk2∆ mutant cells exhibited delayed Htt103QP degradation even in the absence of MG132. The stabilization of Htt103QP was additive in dsk2Δ cells treated with MG132 (Figure 5B). These results support the possibility that Dsk2 may promote Htt103QP clearance through a proteasome-independent mechanism.
Dsk2 promotes Htt103QP clearance through the autophagy pathway
Misfolded proteins are prone to aggregation, and the aggregates are resistant to proteasome-dependent proteolysis, but they can be delivered to lysosomes/vacuoles for disposal (Ciechanover and Kwon, 2015). To test this possibility for Htt103QP, we constructed a VPH1-mApple strain, as Vph1 is a vacuolar membrane protein (Toulmay and Prinz, 2013). The fluorescence signal indicates the vacuole membrane localization of Vph1-mApple, but VPH1-mApple cells expressing Htt103QP showed a low frequency of mApple-GFP colocalization, which could be due to the vigorous degradation of Htt103QP-GFP inside the vacuole. Therefore we examined Htt103QP-GFP localization in pep4Δ VPH1-mApple cells, which lack the key vacuole protease Pep4 (Ammerer ). The cells were grown in galactose medium for 12 h to induce Htt103QP expression, and the mApple and GFP signals were examined after addition of glucose and HU for 2 h. Strikingly, >90% of cells exhibited colocalization of GFP with the vacuole (mApple; Figure 6A). Vph1 exhibited the ring-like structure in WT cells described previously, but we found a homogeneous distribution of Vph1 inside the vacuole in pep4Δ cells. We speculate that vacuole membrane localization of Vph1 is dynamic, and Vph1 is degraded by the hydrolase Pep4 inside the vacuole; thus deletion of the PEP4 gene leads to vacuolar Vph1 accumulation as for other vacuolar membrane proteins (Li ).
FIGURE 6:
The delivery of Htt103QP into vacuoles is autophagy dependent. (A) The vacuolar localization of Htt103QP-GFP in WT and atg1Δ and atg8Δ mutants. pep4Δ, atg1Δ pep4Δ, and atg8Δ pep4Δ cells expressing Vph1-mApple and Htt103QP were grown in galactose medium for 12 h, and then glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. Two hours later, the cells were collected to examine GFP and mApple signals. Representative cell images are shown for the localization of Htt103QP-GFP and Vph1-mApple. Right, percentage of GFP-mApple colocalization (n > 100). (B) Quantitative intensity analysis. Statistical dot plot of the intensity of GFP and mApple within the vacuole of WT and atg1Δ, and atg8Δ mutant cells (n = 20), as well as the medians (black bars). The p values are from the statistical comparison between WT and each mutant.
The delivery of Htt103QP into vacuoles is autophagy dependent. (A) The vacuolar localization of Htt103QP-GFP in WT and atg1Δ and atg8Δ mutants. pep4Δ, atg1Δ pep4Δ, and atg8Δ pep4Δ cells expressing Vph1-mApple and Htt103QP were grown in galactose medium for 12 h, and then glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. Two hours later, the cells were collected to examine GFP and mApple signals. Representative cell images are shown for the localization of Htt103QP-GFP and Vph1-mApple. Right, percentage of GFP-mApple colocalization (n > 100). (B) Quantitative intensity analysis. Statistical dot plot of the intensity of GFP and mApple within the vacuole of WT and atg1Δ, and atg8Δ mutant cells (n = 20), as well as the medians (black bars). The p values are from the statistical comparison between WT and each mutant.The autophagy pathway is responsible for the clearance of misfolded proteins by delivering them to lysosomes/vacuoles for degradation. In this pathway, Atg8 is a ubiquitin-like protein that is anchored to the membrane of autophagosomes and likely mediates membrane fusion with the vacuole, whereas Atg1 is required for autophagy by promoting phagophore assembly (Wen and Klionsky, 2016). To test whether the vacuolar localization of Htt103QP is dependent on the autophagy pathway, we examined the vacuolar localization of Htt103QP-GFP in atg1Δ and atg8Δ cells. Strikingly, very few mutant cells showed obvious vacuolar GFP localization (Figure 6A). Quantitative analysis also indicated a much weaker vacuolar GFP signal in atg1Δ and atg8Δ cells than in WT cells (Figure 6B). Therefore the autophagy pathway is required for the delivery of Htt103QP proteins into the vacuole, supporting the conclusion that Htt103QP is subject to autophagy-dependent disposal after IB formation.To determine the role of Dsk2 in vacuole-dependent disposal of Htt103QP, we compared the vacuolar localization of Htt103QP in pep4Δ and dsk2Δ pep4Δ cells. As described earlier, the cells were grown in galactose medium for 12 h. After addition of glucose and HU for 2 and 4 h, the cells were collected for fluorescence microscopy. At the 2-h time point, 95% of pep4Δ and 71% of dsk2Δ pep4Δ cells showed vacuolar GFP signal. At the 4-h time point, 83% of pep4Δ and 60% of dsk2Δ pep4Δ cells exhibited obvious vacuolar GFP signal (Figure 7A). Consistently, more dsk2Δ cells showed cytoplasmic GFP aggregates without noticeable vacuolar GFP signal compared with WT cells. In addition, the vacuolar GFP signal in dsk2∆ mutants was much weaker than that in WT cells, as evidenced by the quantitative intensity of GFP and mApple signals (Figure 7B). Therefore the delivery of Htt103QP-GFP into vacuoles is compromised in dsk2∆ mutant cells. We speculate that the IB formation defect in dsk2Δ mutants contributes to the failure of autophagy-dependent vacuole delivery of Htt103QP.
FIGURE 7:
The delivery of Htt103QP into vacuoles is compromised in dsk2Δ mutants. pep4Δ VPH1-mApple and dsk2Δ pep4Δ VPH1-mApple cells with Htt103QP plasmid were grown in galactose medium for 12 h, and then glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. The cells were collected at 2 and 4 h after glucose addition to examine the GFP and mApple signals. (A) Representative cell images for the localization of Htt103QP-GFP and Vph1-mApple (2 h), as well as the percentage of the colocalization (n > 100). (B) Statistical dot plot of the intensity of GFP and mApple signals within the vacuole of WT and dsk2Δ mutant cells (n = 20) and medians (black bars). The p values are from the statistical comparison between WT and dsk2Δ cells.
The delivery of Htt103QP into vacuoles is compromised in dsk2Δ mutants. pep4Δ VPH1-mApple and dsk2Δ pep4Δ VPH1-mApple cells with Htt103QP plasmid were grown in galactose medium for 12 h, and then glucose (2%) and HU (200 mM) were added into the medium to shut off Htt103QP expression and block cell cycle progression. The cells were collected at 2 and 4 h after glucose addition to examine the GFP and mApple signals. (A) Representative cell images for the localization of Htt103QP-GFP and Vph1-mApple (2 h), as well as the percentage of the colocalization (n > 100). (B) Statistical dot plot of the intensity of GFP and mApple signals within the vacuole of WT and dsk2Δ mutant cells (n = 20) and medians (black bars). The p values are from the statistical comparison between WT and dsk2Δ cells.
Expression of human ubiquilin 1 and 2 restores Htt103QP inclusion body formation in dsk2Δ mutants
Humanubiquilin 1 and 2 are the yeastDsk2 homologues and also contain UBL, STI, and UBA domains (Elsasser and Finley, 2005; Lowe ; Figure 8A). Mutations in the two ubiquilin genes are linked to neurodegenerative diseases (Takalo ). To determine whether ubiquilin 1 and 2 have a conserved function like Dsk2 in IB formation, we constructed yeast-expressing plasmids for humanubiquilin 1 and 2 and then introduced them into dsk2Δ mutants. We found that expression of ubiquilin 1 and 2 suppressed the growth and IB formation defects in dsk2Δ mutants overexpressing Htt103QP (Figure 8, B and C). In addition, more Htt103QP proteins were detected in the pellet fraction in dsk2Δ mutant cells with the ubiquilin plasmids than in dsk2Δ mutant cells (Figure 8D). These results indicate that the function of ubiquilin proteins in IB formation is conserved from yeast to human.
FIGURE 8:
Human ubiquilin 1 and 2 suppress the phenotype of dsk2Δ mutants. (A) Schematic illustration of the domains in yeast Dsk2 and human ubiquilin (UBQLN) proteins. (B) Human ubiquilin 1 or ubiquilin 2 rescues the growth defect of dsk2Δ mutants expressing Htt103QP. WT and dsk2Δ and dsk2Δ mutants with plasmids containing human ubiquilin genes were grown to saturation, 10-fold diluted, and spotted onto synthetic glucose or galactose plates. The plates were incubated at 30°C for 2 d. (C) Human ubiquilin 1 or 2 restores Htt103QP IB formation in dsk2Δ mutants. The same strains were grown in synthetic medium containing galactose for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in representative cells. Bottom, average percentage of cells with different patterns of GFP distribution from three separate experiments (n > 100). Scale bar, 5 μm. (D) Human ubiquilin 1 or 2 restores Htt103QP distribution in detergent-soluble/insoluble factions in dsk2Δ mutants. The same yeast strains were grown in galactose medium for 12 h at 30°C, and the cell lysates were centrifuged and separated into supernatant and pellet fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control.
Humanubiquilin 1 and 2 suppress the phenotype of dsk2Δ mutants. (A) Schematic illustration of the domains in yeastDsk2 and human ubiquilin (UBQLN) proteins. (B) Humanubiquilin 1 or ubiquilin 2 rescues the growth defect of dsk2Δ mutants expressing Htt103QP. WT and dsk2Δ and dsk2Δ mutants with plasmids containing human ubiquilin genes were grown to saturation, 10-fold diluted, and spotted onto synthetic glucose or galactose plates. The plates were incubated at 30°C for 2 d. (C) Humanubiquilin 1 or 2 restores Htt103QP IB formation in dsk2Δ mutants. The same strains were grown in synthetic medium containing galactose for 12 h at 30°C to induce Htt103QP expression. Top, GFP signal in representative cells. Bottom, average percentage of cells with different patterns of GFP distribution from three separate experiments (n > 100). Scale bar, 5 μm. (D) Humanubiquilin 1 or 2 restores Htt103QP distribution in detergent-soluble/insoluble factions in dsk2Δ mutants. The same yeast strains were grown in galactose medium for 12 h at 30°C, and the cell lysates were centrifuged and separated into supernatant and pellet fractions. Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control.
DISCUSSION
In addition to proteasome-mediated protein degradation, lysosomes/vacuoles play a critical role in the clearance of misfolded proteins. However, it is not fully understood how misfolded proteins are packaged and delivered into lysosomes. Using misfolded Htt103QP as a model substrate, we demonstrated the role of yeast ubiquilin Dsk2 in IB formation. Surprisingly, Dsk2 is not required for Htt103QP degradation after a short induction before noticeable IB formation, but the clearance of Htt103QP after a long induction is impaired in dsk2Δ mutants, presumably due to less efficient IB formation. We observed vacuolar localization of Htt103QP that is dependent on the autophagy pathway, but dsk2∆ mutant cells showed significantly compromised vacuolar localization of Htt103QP. Therefore our data from budding yeast suggest that ubiquilin proteins promote autophagy-mediated clearance of misfolded proteins by facilitating IB formation. The suppression of the IB formation defect in dsk2Δ mutants by the expression of human ubiquilin proteins indicates the functional conservation of this protein family.Ubiquilin proteins have been implicated in the pathogenesis of numerous neurodegenerative diseases (Takalo ). It appears that ubiquilin proteins play a critical role in maintaining proteostasis, but the mechanism remains elusive. The observation of delayed degradation of a proteasome substrate in ubiquilin 2 mutant cells supports the possibility that ubiquilin delivers ubiquitinated substrates to proteasome for degradation (Deng ). However, we found that, after a short induction, the degradation of Htt103QP in dsk2Δ mutants is as efficient as in WT cells. We reason that ubiquilin/Dsk2 is dispensable for proteasome-mediated degradation of Htt103QP. In contrast, deletion of another UBL-UBA–encoding gene, RAD23, led to delayed degradation of Htt103QP after a short induction, suggesting that Rad23 likely transports ubiquitinated Htt103QP to proteasomes for degradation.The UBA domain of Dsk2 protein is likely responsible for the interaction with ubiquitinated proteins (Ohno ). The interaction of Htt103QP with the three UBL-UBA proteins indicates that Htt103QP is likely ubiquitinated in budding yeast. Recent work found the ubiquitination of Htt103QP in yeast cells (Yang ). Previous data show that huntingtin is phosphorylated at serine 13 and 16 by an inflammatory kinase IKK in mammalian cells, and this phosphorylation is essential for huntingtin ubiquitination (Thompson ). Because there is no IKK homologue in budding yeast, it is unclear how Htt103QP is ubiquitinated. One possibility is that a yeast kinase phosphorylates Htt103QP to allow its ubiquitination. Alternatively, Htt103QP could be ubiquitinated in budding yeast without phosphorylation, but further experiments are needed to test these possibilities.The three UBL-UBA proteins in budding yeast are Dsk2, Rad23, and Ddi1. Although all three proteins show similar binding affinity to Htt103QP, only Dsk2 is required for Htt103QP IB formation. Moreover, expression of chimeric proteins containing the Dsk2 UBL domain and other domains of Rad23 or Ddi1 is able to promote IB formation. Therefore the UBL but not the UBA domain of Dsk2 contributes to its functional specificity in IB formation. It is possible that a unique interaction between the Dsk2 UBL domain and other proteins is responsible for this specificity. One candidate is the proteasome subunit Rpn10, an intrinsic ubiquitin receptor. The UBL domain of Dsk2 shows specific interaction with Rpn10. Unlike other proteasome proteins, Rpn10 is present in proteasome-bound and free forms. Previous work shows that overexpression of Dsk2 is toxic to yeast cells and results in increased accumulation of ubiquitin conjugates, but the effects of Dsk2 overexpression are alleviated by Rpn10 overexpression (Matiuhin ), most likely due to extraproteasomal Rpn10, which restricts Dsk2’s access to the proteasome (Walters and Zhang, 2008). Therefore the nonproteasomal Rpn10 may interact with Dsk2 to facilitate IB formation. However, we cannot exclude the possibility that the interaction of Dsk2 with proteasomal Rpn10 facilitates the degradation of other protein substrates but does not play a role in Htt103QP IB formation.The colocalization of ubiquilin with autophagosome cargo protein p62/SQSTM indicates the potential role of ubiquilin in autophagy-mediated clearance of misfolded proteins (Ceballos-Diaz ; Osaka ). Using budding yeast as a model organism, we show that ubiquilin Dsk2 promotes the formation of Htt103QP IBs. Moreover, we demonstrate the essential role of the autophagy pathway in the delivery of Htt103QP into the vacuole, and this delivery process is significantly impaired in dsk2Δ cells. These results support the conclusion that Dsk2 promotes the clearance of Htt103QP through the autophagy pathway. However, the link between ubiquilin proteins and the autophagy pathway remains unclear. The ubiquitin-binding protein Cue5 was shown to mediate the clearance of polyQ proteins through autophagy (Lu , b). It will be of interest to test the Cue5-Dsk2 interaction and investigate the role of ubiquilin/Dsk2 in the recruitment of misfolded proteins to the autophagy machinery. Together our results demonstrate that ubiquilin proteins promote IB formation and facilitate the clearance of mutated Htt through the autophagy pathway. Moreover, this mechanism is conserved from yeast to human cells.
MATERIALS AND METHODS
Strains, plasmids, and growth conditions
All of the yeast strains used in this study are isogenic to Y300, a W303 derivative. The relevant genotypes are listed in Supplemental Table S1. Gene deletions and epitope tagging were performed using a PCR-based protocol (Longtine ). The 13×Myc-tagged Dsk2, Rad23, and Ddi1, dsk2∆ and dsk2∆ domain-deletion mutants, and UBL chimeras were confirmed with PCR. The Flag- and GFP-tagged Htt103QP fragment with galactose-inducible promoter (P) was originally from the Lindquist lab (Duennwald ) and integrated into yeast genome. Ubiquilin 1 and 2–expressing plasmids were constructed by inserting the gene fragment of humanubiquilin 1 and 2 into a pRS415 vector. The plasmids p4458 FLAG-hPLIC-1 (ubiquilin 1) and p4455 FLAG-hPLIC-2 (ubiquilin 2), were originally from the Howley lab (Harvard Medical School; plasmids 8663 and 8661; Addgene, Cambridge, MA). Yeast extract/peptone medium supplied with raffinose, glucose, or galactose was used for the growth of yeast strains, except for those carrying plasmids.
Fluorescence image analysis
The analysis of Htt103QP IB formation in fixed cells was carried out using a fluorescence microscope (EVOS; Thermo Fisher Scientific, Waltham, MA). The cells were collected and fixed with 4% paraformaldehyde for 10 min at room temperature. Fluorescence signals from these cells were examined under a fluorescence microscope with a 60× objective.
Western blotting
Protein samples were prepared using an alkaline method and resolved by 10% SDS–PAGE. Anti-Myc antibody was purchased from Covance (Madison, WI); anti-Flag antibody was from Sigma-Aldrich (St. Louis, MO); anti-GFP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-Pgk1 antibody was from Molecular Probes (Eugene, OR). The horseradish peroxidase–conjugated goat anti-mouse immunoglobulin G secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA).
Protein fractionation assay
Cells expressing Htt103QP were collected and resuspended in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 25 mM EDTA, 0.2% [vol/vol] Triton X-100) supplemented with protease inhibitors and phenylmethylsulfonyl fluoride and then broken using a bead beater. The lysates were centrifuged at 14,000 rpm for 30 min at 4°C to separate supernatant and pellet fractions, and the pellet was resuspended in 1× loading buffer (equal volume as the supernatant). Equal volumes of supernatant and pellet fractions were loaded and subject to Western blot analysis.
Coimmunoprecipitation assay
Cell cultures growing in galactose at 30°C for 4 h (to induce Htt103QP expression) were collected and washed once with water. After being resuspended in RIPA buffer (25 mM Tris, pH 7.5, 10 mM EDTA, 150 mM NaCl, and 0.05% Tween-20) supplied with protease inhibitors, cells were broken with a bead beater. The resulting cell extracts were incubated with primary antibody overnight at 4°C. The cell extracts were then incubated with protein A/G PLUS agarose beads (Santa Cruz Biotechnology) for 2 h at room temperature. After incubation, the beads were collected by centrifugation and washed three times with RIPA buffer supplied with protease inhibitors. After removal of RIPA buffer, protein loading buffer was added, and the protein samples were boiled for 5 min for Western blotting.
Statistical analysis
Experimental data are expressed as mean ± SEM. The distribution of fluorescence intensity is compared between WT cells and dsk2Δ mutants by using the Mann–Whitney test (two tailed) and between WT cells and atg1Δ and atg8Δ mutants by using one-way analysis of variance, followed by a Dunnett’s multiple comparison test to compare the difference between each of the mutants and WT cells. Differences with p < 0.05 are considered statistically significant.
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Paolo Bonaldo; Srinivasa Reddy Bonam; Laura Bonfili; Juan S Bonifacino; Brian A Boone; Martin D Bootman; Matteo Bordi; Christoph Borner; Beat C Bornhauser; Gautam Borthakur; Jürgen Bosch; Santanu Bose; Luis M Botana; Juan Botas; Chantal M Boulanger; Michael E Boulton; Mathieu Bourdenx; Benjamin Bourgeois; Nollaig M Bourke; Guilhem Bousquet; Patricia Boya; Peter V Bozhkov; Luiz H M Bozi; Tolga O Bozkurt; Doug E Brackney; Christian H Brandts; Ralf J Braun; Gerhard H Braus; Roberto Bravo-Sagua; José M Bravo-San Pedro; Patrick Brest; Marie-Agnès Bringer; Alfredo Briones-Herrera; V Courtney Broaddus; Peter Brodersen; Jeffrey L Brodsky; Steven L Brody; Paola G Bronson; Jeff M Bronstein; Carolyn N Brown; Rhoderick E Brown; Patricia C Brum; John H Brumell; Nicola Brunetti-Pierri; Daniele Bruno; Robert J Bryson-Richardson; Cecilia Bucci; Carmen Buchrieser; Marta Bueno; Laura Elisa Buitrago-Molina; Simone Buraschi; Shilpa Buch; J Ross Buchan; Erin M Buckingham; Hikmet Budak; Mauricio Budini; Geert Bultynck; Florin Burada; Joseph R Burgoyne; M Isabel Burón; Victor Bustos; Sabrina Büttner; Elena Butturini; Aaron Byrd; Isabel Cabas; Sandra Cabrera-Benitez; Ken Cadwell; Jingjing Cai; Lu Cai; Qian Cai; Montserrat Cairó; Jose A Calbet; Guy A Caldwell; Kim A Caldwell; Jarrod A Call; Riccardo Calvani; Ana C Calvo; Miguel Calvo-Rubio Barrera; Niels Os Camara; Jacques H Camonis; Nadine Camougrand; Michelangelo Campanella; Edward M Campbell; François-Xavier Campbell-Valois; Silvia Campello; Ilaria Campesi; Juliane C Campos; Olivier Camuzard; Jorge Cancino; Danilo Candido de Almeida; Laura Canesi; Isabella Caniggia; Barbara Canonico; Carles Cantí; Bin Cao; Michele Caraglia; Beatriz Caramés; Evie H Carchman; Elena Cardenal-Muñoz; Cesar Cardenas; Luis Cardenas; Sandra M Cardoso; Jennifer S Carew; Georges F Carle; Gillian Carleton; Silvia Carloni; Didac Carmona-Gutierrez; Leticia A Carneiro; Oliana Carnevali; Julian M Carosi; Serena Carra; Alice Carrier; Lucie Carrier; Bernadette Carroll; A Brent Carter; Andreia Neves Carvalho; Magali Casanova; Caty Casas; Josefina Casas; Chiara Cassioli; Eliseo F Castillo; Karen Castillo; Sonia Castillo-Lluva; Francesca Castoldi; Marco Castori; Ariel F Castro; Margarida Castro-Caldas; Javier Castro-Hernandez; Susana Castro-Obregon; Sergio D Catz; Claudia Cavadas; Federica Cavaliere; Gabriella Cavallini; Maria Cavinato; Maria L Cayuela; Paula Cebollada Rica; Valentina Cecarini; Francesco Cecconi; Marzanna Cechowska-Pasko; Simone Cenci; Victòria Ceperuelo-Mallafré; João J Cerqueira; Janete M Cerutti; Davide Cervia; Vildan Bozok Cetintas; Silvia Cetrullo; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Oishee Chakrabarti; Tapas Chakraborty; Trinad Chakraborty; Mounia Chami; Georgios Chamilos; David W Chan; Edmond Y W Chan; Edward D Chan; H Y Edwin Chan; Helen H Chan; Hung Chan; Matthew T V Chan; Yau Sang Chan; Partha K Chandra; Chih-Peng Chang; Chunmei Chang; Hao-Chun Chang; Kai Chang; Jie Chao; Tracey Chapman; Nicolas Charlet-Berguerand; Samrat Chatterjee; Shail K Chaube; Anu Chaudhary; Santosh Chauhan; Edward Chaum; Frédéric Checler; Michael E Cheetham; Chang-Shi Chen; Guang-Chao Chen; Jian-Fu Chen; Liam L Chen; Leilei Chen; Lin Chen; Mingliang Chen; Mu-Kuan Chen; Ning Chen; Quan Chen; Ruey-Hwa Chen; Shi Chen; Wei Chen; Weiqiang Chen; Xin-Ming Chen; Xiong-Wen Chen; Xu Chen; Yan Chen; Ye-Guang Chen; Yingyu Chen; Yongqiang Chen; Yu-Jen Chen; Yue-Qin Chen; Zhefan Stephen Chen; Zhi Chen; Zhi-Hua Chen; Zhijian J Chen; Zhixiang Chen; Hanhua Cheng; Jun Cheng; Shi-Yuan Cheng; Wei Cheng; Xiaodong Cheng; Xiu-Tang Cheng; Yiyun Cheng; Zhiyong Cheng; Zhong Chen; Heesun Cheong; Jit Kong Cheong; Boris V Chernyak; Sara Cherry; Chi Fai Randy Cheung; Chun Hei Antonio Cheung; King-Ho Cheung; Eric Chevet; Richard J Chi; Alan Kwok Shing Chiang; Ferdinando Chiaradonna; Roberto Chiarelli; Mario Chiariello; Nathalia Chica; Susanna Chiocca; Mario Chiong; Shih-Hwa Chiou; Abhilash I Chiramel; Valerio Chiurchiù; Dong-Hyung Cho; Seong-Kyu Choe; Augustine M K Choi; Mary E Choi; Kamalika Roy Choudhury; Norman S Chow; Charleen T Chu; Jason P Chua; John Jia En Chua; Hyewon Chung; Kin Pan Chung; Seockhoon Chung; So-Hyang Chung; Yuen-Li Chung; Valentina Cianfanelli; Iwona A Ciechomska; Mariana Cifuentes; Laura Cinque; Sebahattin Cirak; Mara Cirone; Michael J Clague; Robert Clarke; Emilio Clementi; Eliana M Coccia; Patrice Codogno; Ehud Cohen; Mickael M Cohen; Tania Colasanti; Fiorella Colasuonno; Robert A Colbert; Anna Colell; Miodrag Čolić; Nuria S Coll; Mark O Collins; María I Colombo; Daniel A Colón-Ramos; Lydie Combaret; Sergio Comincini; Márcia R Cominetti; Antonella Consiglio; Andrea Conte; Fabrizio Conti; Viorica Raluca Contu; Mark R Cookson; Kevin M Coombs; Isabelle Coppens; Maria Tiziana Corasaniti; Dale P Corkery; Nils Cordes; Katia Cortese; Maria do Carmo Costa; Sarah Costantino; Paola Costelli; Ana Coto-Montes; Peter J Crack; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Riccardo Cristofani; Tamas Csizmadia; Antonio Cuadrado; Bing Cui; Jun Cui; Yixian Cui; Yong Cui; Emmanuel Culetto; Andrea C Cumino; Andrey V Cybulsky; Mark J Czaja; Stanislaw J Czuczwar; Stefania D'Adamo; Marcello D'Amelio; Daniela D'Arcangelo; Andrew C D'Lugos; Gabriella D'Orazi; James A da Silva; Hormos Salimi Dafsari; Ruben K Dagda; Yasin Dagdas; Maria Daglia; Xiaoxia Dai; Yun Dai; Yuyuan Dai; Jessica Dal Col; Paul Dalhaimer; Luisa Dalla Valle; Tobias Dallenga; Guillaume Dalmasso; Markus Damme; Ilaria Dando; Nico P Dantuma; April L Darling; Hiranmoy Das; Srinivasan Dasarathy; Santosh K Dasari; Srikanta Dash; Oliver Daumke; Adrian N Dauphinee; Jeffrey S Davies; Valeria A Dávila; Roger J Davis; Tanja Davis; Sharadha Dayalan Naidu; Francesca De Amicis; Karolien De Bosscher; Francesca De Felice; Lucia De Franceschi; Chiara De Leonibus; Mayara G de Mattos Barbosa; Guido R Y De Meyer; Angelo De Milito; Cosimo De Nunzio; Clara De Palma; Mauro De Santi; Claudio De Virgilio; Daniela De Zio; Jayanta Debnath; Brian J DeBosch; Jean-Paul Decuypere; Mark A Deehan; Gianluca Deflorian; James DeGregori; Benjamin Dehay; Gabriel Del Rio; Joe R Delaney; Lea M D Delbridge; Elizabeth Delorme-Axford; M Victoria Delpino; Francesca Demarchi; Vilma Dembitz; Nicholas D Demers; Hongbin Deng; Zhiqiang Deng; Joern Dengjel; Paul Dent; Donna Denton; Melvin L DePamphilis; Channing J Der; Vojo Deretic; Albert Descoteaux; Laura Devis; Sushil Devkota; Olivier Devuyst; Grant Dewson; Mahendiran Dharmasivam; Rohan Dhiman; Diego di Bernardo; Manlio Di Cristina; Fabio Di Domenico; Pietro Di Fazio; Alessio Di Fonzo; Giovanni Di Guardo; Gianni M Di Guglielmo; Luca Di Leo; Chiara Di Malta; Alessia Di Nardo; Martina Di Rienzo; Federica Di Sano; George Diallinas; Jiajie Diao; Guillermo Diaz-Araya; Inés Díaz-Laviada; Jared M Dickinson; Marc Diederich; Mélanie Dieudé; Ivan Dikic; Shiping Ding; Wen-Xing Ding; Luciana Dini; Jelena Dinić; Miroslav Dinic; Albena T Dinkova-Kostova; Marc S Dionne; Jörg H W Distler; Abhinav Diwan; Ian M C Dixon; Mojgan Djavaheri-Mergny; Ina Dobrinski; Oxana Dobrovinskaya; Radek Dobrowolski; Renwick C J Dobson; Jelena Đokić; Serap Dokmeci Emre; Massimo Donadelli; Bo Dong; Xiaonan Dong; Zhiwu Dong; Gerald W Dorn Ii; Volker Dotsch; Huan Dou; Juan Dou; Moataz Dowaidar; Sami Dridi; Liat Drucker; Ailian Du; Caigan Du; Guangwei Du; Hai-Ning Du; Li-Lin Du; André du Toit; Shao-Bin Duan; Xiaoqiong Duan; Sónia P Duarte; Anna Dubrovska; Elaine A Dunlop; Nicolas Dupont; Raúl V Durán; Bilikere S Dwarakanath; Sergey A Dyshlovoy; Darius Ebrahimi-Fakhari; Leopold Eckhart; Charles L Edelstein; Thomas Efferth; Eftekhar Eftekharpour; Ludwig Eichinger; Nabil Eid; Tobias Eisenberg; N Tony Eissa; Sanaa Eissa; Miriam Ejarque; Abdeljabar El Andaloussi; Nazira El-Hage; Shahenda El-Naggar; Anna Maria Eleuteri; Eman S El-Shafey; Mohamed Elgendy; Aristides G Eliopoulos; María M Elizalde; Philip M Elks; Hans-Peter Elsasser; Eslam S Elsherbiny; Brooke M Emerling; N C Tolga Emre; Christina H Eng; Nikolai Engedal; Anna-Mart Engelbrecht; Agnete S T Engelsen; Jorrit M Enserink; Ricardo Escalante; Audrey Esclatine; Mafalda Escobar-Henriques; Eeva-Liisa Eskelinen; Lucile Espert; Makandjou-Ola Eusebio; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Francesco Facchiano; Bengt Fadeel; Claudio Fader; Alex C Faesen; W Douglas Fairlie; Alberto Falcó; Bjorn H Falkenburger; Daping Fan; Jie Fan; Yanbo Fan; Evandro F Fang; Yanshan Fang; Yognqi Fang; Manolis Fanto; Tamar Farfel-Becker; Mathias Faure; Gholamreza Fazeli; Anthony O Fedele; Arthur M Feldman; Du Feng; Jiachun Feng; Lifeng Feng; Yibin Feng; Yuchen Feng; Wei Feng; Thais Fenz Araujo; Thomas A Ferguson; Álvaro F Fernández; Jose C Fernandez-Checa; Sonia Fernández-Veledo; Alisdair R Fernie; Anthony W Ferrante; Alessandra Ferraresi; Merari F Ferrari; Julio C B Ferreira; Susan Ferro-Novick; Antonio Figueras; Riccardo Filadi; Nicoletta Filigheddu; Eduardo Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; Vittorio Fineschi; Francesca Finetti; Steven Finkbeiner; Edward A Fisher; Paul B Fisher; Flavio Flamigni; Steven J Fliesler; Trude H Flo; Ida Florance; Oliver Florey; Tullio Florio; Erika Fodor; Carlo Follo; Edward A Fon; Antonella Forlino; Francesco Fornai; Paola Fortini; Anna Fracassi; Alessandro Fraldi; Brunella Franco; Rodrigo Franco; Flavia Franconi; Lisa B Frankel; Scott L Friedman; Leopold F Fröhlich; Gema Frühbeck; Jose M Fuentes; Yukio Fujiki; Naonobu Fujita; Yuuki Fujiwara; Mitsunori Fukuda; Simone Fulda; Luc Furic; Norihiko Furuya; Carmela Fusco; Michaela U Gack; Lidia Gaffke; Sehamuddin Galadari; Alessia Galasso; Maria F Galindo; Sachith Gallolu Kankanamalage; Lorenzo Galluzzi; Vincent Galy; Noor Gammoh; Boyi Gan; Ian G Ganley; Feng Gao; Hui Gao; Minghui Gao; Ping Gao; Shou-Jiang Gao; Wentao Gao; Xiaobo Gao; Ana Garcera; Maria Noé Garcia; Verónica E Garcia; Francisco García-Del Portillo; Vega Garcia-Escudero; Aracely Garcia-Garcia; Marina Garcia-Macia; Diana García-Moreno; Carmen Garcia-Ruiz; Patricia García-Sanz; Abhishek D Garg; Ricardo Gargini; Tina Garofalo; Robert F Garry; Nils C Gassen; Damian Gatica; Liang Ge; Wanzhong Ge; Ruth Geiss-Friedlander; Cecilia Gelfi; Pascal Genschik; Ian E Gentle; Valeria Gerbino; Christoph Gerhardt; Kyla Germain; Marc Germain; David A Gewirtz; Elham Ghasemipour Afshar; Saeid Ghavami; Alessandra Ghigo; Manosij Ghosh; Georgios Giamas; Claudia Giampietri; Alexandra Giatromanolaki; Gary E Gibson; Spencer B Gibson; Vanessa Ginet; Edward Giniger; Carlotta Giorgi; Henrique Girao; Stephen E Girardin; Mridhula Giridharan; Sandy Giuliano; Cecilia Giulivi; Sylvie Giuriato; Julien Giustiniani; Alexander Gluschko; Veit Goder; Alexander Goginashvili; Jakub Golab; David C Goldstone; Anna Golebiewska; Luciana R Gomes; Rodrigo Gomez; Rubén Gómez-Sánchez; Maria Catalina Gomez-Puerto; Raquel Gomez-Sintes; Qingqiu Gong; Felix M Goni; Javier González-Gallego; Tomas Gonzalez-Hernandez; Rosa A Gonzalez-Polo; Jose A Gonzalez-Reyes; Patricia González-Rodríguez; Ing Swie Goping; Marina S Gorbatyuk; Nikolai V Gorbunov; Kıvanç Görgülü; Roxana M Gorojod; Sharon M Gorski; Sandro Goruppi; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Martin Graef; Markus H Gräler; Veronica Granatiero; Daniel Grasso; Joshua P Gray; Douglas R Green; Alexander Greenhough; Stephen L Gregory; Edward F Griffin; Mark W Grinstaff; Frederic Gros; Charles Grose; Angelina S Gross; Florian Gruber; Paolo Grumati; Tilman Grune; Xueyan Gu; Jun-Lin Guan; Carlos M Guardia; Kishore Guda; Flora Guerra; Consuelo Guerri; Prasun Guha; Carlos Guillén; Shashi Gujar; Anna Gukovskaya; Ilya Gukovsky; Jan Gunst; Andreas Günther; Anyonya R Guntur; Chuanyong Guo; Chun Guo; Hongqing Guo; Lian-Wang Guo; Ming Guo; Pawan Gupta; Shashi Kumar Gupta; Swapnil Gupta; Veer Bala Gupta; Vivek Gupta; Asa B Gustafsson; David D Gutterman; Ranjitha H B; Annakaisa Haapasalo; James E Haber; Aleksandra Hać; Shinji Hadano; Anders J Hafrén; Mansour Haidar; Belinda S Hall; Gunnel Halldén; Anne Hamacher-Brady; Andrea Hamann; Maho Hamasaki; Weidong Han; Malene Hansen; Phyllis I Hanson; Zijian Hao; Masaru Harada; Ljubica Harhaji-Trajkovic; Nirmala Hariharan; Nigil Haroon; James Harris; Takafumi Hasegawa; Noor Hasima Nagoor; Jeffrey A Haspel; Volker Haucke; Wayne D Hawkins; Bruce A Hay; Cole M Haynes; Soren B Hayrabedyan; Thomas S Hays; Congcong He; Qin He; Rong-Rong He; You-Wen He; Yu-Ying He; Yasser Heakal; Alexander M Heberle; J Fielding Hejtmancik; Gudmundur Vignir Helgason; Vanessa Henkel; Marc Herb; Alexander Hergovich; Anna Herman-Antosiewicz; Agustín Hernández; Carlos Hernandez; Sergio Hernandez-Diaz; Virginia Hernandez-Gea; Amaury Herpin; Judit Herreros; Javier H Hervás; Daniel Hesselson; Claudio Hetz; Volker T Heussler; Yujiro Higuchi; Sabine Hilfiker; Joseph A Hill; William S Hlavacek; Emmanuel A Ho; Idy H T Ho; Philip Wing-Lok Ho; Shu-Leong Ho; Wan Yun Ho; G Aaron Hobbs; Mark Hochstrasser; Peter H M Hoet; Daniel Hofius; Paul Hofman; Annika Höhn; Carina I Holmberg; Jose R Hombrebueno; Chang-Won Hong Yi-Ren Hong; Lora V Hooper; Thorsten Hoppe; Rastislav Horos; Yujin Hoshida; I-Lun Hsin; Hsin-Yun Hsu; Bing Hu; Dong Hu; Li-Fang Hu; Ming Chang Hu; Ronggui Hu; Wei Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Jinlian Hua; Yingqi Hua; Chongmin Huan; Canhua Huang; Chuanshu Huang; Chuanxin Huang; Chunling Huang; Haishan Huang; Kun Huang; Michael L H Huang; Rui Huang; Shan Huang; Tianzhi Huang; Xing Huang; Yuxiang Jack Huang; Tobias B Huber; Virginie Hubert; Christian A Hubner; Stephanie M Hughes; William E Hughes; Magali Humbert; Gerhard Hummer; James H Hurley; Sabah Hussain; Salik Hussain; Patrick J Hussey; Martina Hutabarat; Hui-Yun Hwang; Seungmin Hwang; Antonio Ieni; Fumiyo Ikeda; Yusuke Imagawa; Yuzuru Imai; Carol Imbriano; Masaya Imoto; Denise M Inman; Ken Inoki; Juan Iovanna; Renato V Iozzo; Giuseppe Ippolito; Javier E Irazoqui; Pablo Iribarren; Mohd Ishaq; Makoto Ishikawa; Nestor Ishimwe; Ciro Isidoro; Nahed Ismail; Shohreh Issazadeh-Navikas; Eisuke Itakura; Daisuke Ito; Davor Ivankovic; Saška Ivanova; Anand Krishnan V Iyer; José M Izquierdo; Masanori Izumi; Marja Jäättelä; Majid Sakhi Jabir; William T Jackson; Nadia Jacobo-Herrera; Anne-Claire Jacomin; Elise Jacquin; Pooja Jadiya; Hartmut Jaeschke; Chinnaswamy Jagannath; Arjen J Jakobi; Johan Jakobsson; Bassam Janji; Pidder Jansen-Dürr; Patric J Jansson; Jonathan Jantsch; Sławomir Januszewski; Alagie Jassey; Steve Jean; Hélène Jeltsch-David; Pavla Jendelova; Andreas Jenny; Thomas E Jensen; Niels Jessen; Jenna L Jewell; Jing Ji; Lijun Jia; Rui Jia; Liwen Jiang; Qing Jiang; Richeng Jiang; Teng Jiang; Xuejun Jiang; Yu Jiang; Maria Jimenez-Sanchez; Eun-Jung Jin; Fengyan Jin; Hongchuan Jin; Li Jin; Luqi Jin; Meiyan Jin; Si Jin; Eun-Kyeong Jo; Carine Joffre; Terje Johansen; Gail V W Johnson; Simon A Johnston; Eija Jokitalo; Mohit Kumar Jolly; Leo A B Joosten; Joaquin Jordan; Bertrand Joseph; Dianwen Ju; Jeong-Sun Ju; Jingfang Ju; Esmeralda Juárez; Delphine Judith; Gábor Juhász; Youngsoo Jun; Chang Hwa Jung; Sung-Chul Jung; Yong Keun Jung; Heinz Jungbluth; Johannes Jungverdorben; Steffen Just; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Daniel Kaganovich; Alon Kahana; Renate Kain; Shinjo Kajimura; Maria Kalamvoki; Manjula Kalia; Danuta S Kalinowski; Nina Kaludercic; Ioanna Kalvari; Joanna Kaminska; Vitaliy O Kaminskyy; Hiromitsu Kanamori; Keizo Kanasaki; Chanhee Kang; Rui Kang; Sang Sun Kang; Senthilvelrajan Kaniyappan; Tomotake Kanki; Thirumala-Devi Kanneganti; Anumantha G Kanthasamy; Arthi Kanthasamy; Marc Kantorow; Orsolya Kapuy; Michalis V Karamouzis; Md Razaul Karim; Parimal Karmakar; Rajesh G Katare; Masaru Kato; Stefan H E Kaufmann; Anu Kauppinen; Gur P Kaushal; Susmita Kaushik; Kiyoshi Kawasaki; Kemal Kazan; Po-Yuan Ke; Damien J Keating; Ursula Keber; John H Kehrl; Kate E Keller; Christian W Keller; Jongsook Kim Kemper; Candia M Kenific; Oliver Kepp; Stephanie Kermorgant; Andreas Kern; Robin Ketteler; Tom G Keulers; Boris Khalfin; Hany Khalil; Bilon Khambu; Shahid Y Khan; Vinoth Kumar Megraj Khandelwal; Rekha Khandia; Widuri Kho; Noopur V Khobrekar; Sataree Khuansuwan; Mukhran Khundadze; Samuel A Killackey; Dasol Kim; Deok Ryong Kim; Do-Hyung Kim; Dong-Eun Kim; Eun Young Kim; Eun-Kyoung Kim; Hak-Rim Kim; Hee-Sik Kim; Jeong Hun Kim; Jin Kyung Kim; Jin-Hoi Kim; Joungmok Kim; Ju Hwan Kim; Keun Il Kim; Peter K Kim; Seong-Jun Kim; Scot R Kimball; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Matthew A King; Kerri J Kinghorn; Conan G Kinsey; Vladimir Kirkin; Lorrie A Kirshenbaum; Sergey L Kiselev; Shuji Kishi; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Richard N Kitsis; Josef T Kittler; Ole Kjaerulff; Peter S Klein; Thomas Klopstock; Jochen Klucken; Helene Knævelsrud; Roland L Knorr; Ben C B Ko; Fred Ko; Jiunn-Liang Ko; Hotaka Kobayashi; Satoru Kobayashi; Ina Koch; Jan C Koch; Ulrich Koenig; Donat Kögel; Young Ho Koh; Masato Koike; Sepp D Kohlwein; Nur M Kocaturk; Masaaki Komatsu; Jeannette König; Toru Kono; Benjamin T Kopp; Tamas Korcsmaros; Gözde Korkmaz; Viktor I Korolchuk; Mónica Suárez Korsnes; Ali Koskela; Janaiah Kota; Yaichiro Kotake; Monica L Kotler; Yanjun Kou; Michael I Koukourakis; Evangelos Koustas; Attila L Kovacs; Tibor Kovács; Daisuke Koya; Tomohiro Kozako; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Anna D Krasnodembskaya; Carole Kretz-Remy; Guido Kroemer; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Sabine Kuenen; Lars Kuerschner; Thomas Kukar; Ajay Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Sharad Kumar; Shinji Kume; Caroline Kumsta; Chanakya N Kundu; Mondira Kundu; Ajaikumar B Kunnumakkara; Lukasz Kurgan; Tatiana G Kutateladze; Ozlem Kutlu; SeongAe Kwak; Ho Jeong Kwon; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert La Spada; Patrick Labonté; Sylvain Ladoire; Ilaria Laface; Frank Lafont; Diane C Lagace; Vikramjit Lahiri; Zhibing Lai; Angela S Laird; Aparna Lakkaraju; Trond Lamark; Sheng-Hui Lan; Ane Landajuela; Darius J R Lane; Jon D Lane; Charles H Lang; Carsten Lange; Ülo Langel; Rupert Langer; Pierre Lapaquette; Jocelyn Laporte; Nicholas F LaRusso; Isabel Lastres-Becker; Wilson Chun Yu Lau; Gordon W Laurie; Sergio Lavandero; Betty Yuen Kwan Law; Helen Ka-Wai Law; Rob Layfield; Weidong Le; Herve Le Stunff; Alexandre Y Leary; Jean-Jacques Lebrun; Lionel Y W Leck; Jean-Philippe Leduc-Gaudet; Changwook Lee; Chung-Pei Lee; Da-Hye Lee; Edward B Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Heung Kyu Lee; Jae Man Lee; Jason S Lee; Jin-A Lee; Joo-Yong Lee; Jun Hee Lee; Michael Lee; Min Goo Lee; Min Jae Lee; Myung-Shik Lee; Sang Yoon Lee; Seung-Jae Lee; Stella Y Lee; Sung Bae Lee; Won Hee Lee; Ying-Ray Lee; Yong-Ho Lee; Youngil Lee; Christophe Lefebvre; Renaud Legouis; Yu L Lei; Yuchen Lei; Sergey Leikin; Gerd Leitinger; Leticia Lemus; Shuilong Leng; Olivia Lenoir; Guido Lenz; Heinz Josef Lenz; Paola Lenzi; Yolanda León; Andréia M Leopoldino; Christoph Leschczyk; Stina Leskelä; Elisabeth Letellier; Chi-Ting Leung; Po Sing Leung; Jeremy S Leventhal; Beth Levine; Patrick A Lewis; Klaus Ley; Bin Li; Da-Qiang Li; Jianming Li; Jing Li; Jiong Li; Ke Li; Liwu Li; Mei Li; Min Li; Min Li; Ming Li; Mingchuan Li; Pin-Lan Li; Ming-Qing Li; Qing Li; Sheng Li; Tiangang Li; Wei Li; Wenming Li; Xue Li; Yi-Ping Li; Yuan Li; Zhiqiang Li; Zhiyong Li; Zhiyuan Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Weicheng Liang; Yongheng Liang; YongTian Liang; Guanghong Liao; Lujian Liao; Mingzhi Liao; Yung-Feng Liao; Mariangela Librizzi; Pearl P Y Lie; Mary A Lilly; Hyunjung J Lim; Thania R R Lima; Federica Limana; Chao Lin; Chih-Wen Lin; Dar-Shong Lin; Fu-Cheng Lin; Jiandie D Lin; Kurt M Lin; Kwang-Huei Lin; Liang-Tzung Lin; Pei-Hui Lin; Qiong Lin; Shaofeng Lin; Su-Ju Lin; Wenyu Lin; Xueying Lin; Yao-Xin Lin; Yee-Shin Lin; Rafael Linden; Paula Lindner; Shuo-Chien Ling; Paul Lingor; Amelia K Linnemann; Yih-Cherng Liou; Marta M Lipinski; Saška Lipovšek; Vitor A Lira; Natalia Lisiak; Paloma B Liton; Chao Liu; Ching-Hsuan Liu; Chun-Feng Liu; Cui Hua Liu; Fang Liu; Hao Liu; Hsiao-Sheng Liu; Hua-Feng Liu; Huifang Liu; Jia Liu; Jing Liu; Julia Liu; Leyuan Liu; Longhua Liu; Meilian Liu; Qin Liu; Wei Liu; Wende Liu; Xiao-Hong Liu; Xiaodong Liu; Xingguo Liu; Xu Liu; Xuedong Liu; Yanfen Liu; Yang Liu; Yang Liu; Yueyang Liu; Yule Liu; J Andrew Livingston; Gerard Lizard; Jose M Lizcano; Senka Ljubojevic-Holzer; Matilde E LLeonart; David Llobet-Navàs; Alicia Llorente; Chih Hung Lo; Damián Lobato-Márquez; Qi Long; Yun Chau Long; Ben Loos; Julia A Loos; Manuela G López; Guillermo López-Doménech; José Antonio López-Guerrero; Ana T López-Jiménez; Óscar López-Pérez; Israel López-Valero; Magdalena J Lorenowicz; Mar Lorente; Peter Lorincz; Laura Lossi; Sophie Lotersztajn; Penny E Lovat; Jonathan F Lovell; Alenka Lovy; Péter Lőw; Guang Lu; Haocheng Lu; Jia-Hong Lu; Jin-Jian Lu; Mengji Lu; Shuyan Lu; Alessandro Luciani; John M Lucocq; Paula Ludovico; Micah A Luftig; Morten Luhr; Diego Luis-Ravelo; Julian J Lum; Liany Luna-Dulcey; Anders H Lund; Viktor K Lund; Jan D Lünemann; Patrick Lüningschrör; Honglin Luo; Rongcan Luo; Shouqing Luo; Zhi Luo; Claudio Luparello; Bernhard Lüscher; Luan Luu; Alex Lyakhovich; Konstantin G Lyamzaev; Alf Håkon Lystad; Lyubomyr Lytvynchuk; Alvin C Ma; Changle Ma; Mengxiao Ma; Ning-Fang Ma; Quan-Hong Ma; Xinliang Ma; Yueyun Ma; Zhenyi Ma; Ormond A MacDougald; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; Sandra Maday; Frank Madeo; Muniswamy Madesh; Tobias Madl; Julio Madrigal-Matute; Akiko Maeda; Yasuhiro Maejima; Marta Magarinos; Poornima Mahavadi; Emiliano Maiani; Kenneth Maiese; Panchanan Maiti; Maria Chiara Maiuri; Barbara Majello; Michael B Major; Elena Makareeva; Fayaz Malik; Karthik Mallilankaraman; Walter Malorni; Alina Maloyan; Najiba Mammadova; Gene Chi Wai Man; Federico Manai; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Masoud H Manjili; Ravi Manjithaya; Patricio Manque; Bella B Manshian; Raquel Manzano; Claudia Manzoni; Kai Mao; Cinzia Marchese; Sandrine Marchetti; Anna Maria Marconi; Fabrizio Marcucci; Stefania Mardente; Olga A Mareninova; Marta Margeta; Muriel Mari; Sara Marinelli; Oliviero Marinelli; Guillermo Mariño; Sofia Mariotto; Richard S Marshall; Mark R Marten; Sascha Martens; Alexandre P J Martin; Katie R Martin; Sara Martin; Shaun Martin; Adrián Martín-Segura; Miguel A Martín-Acebes; Inmaculada Martin-Burriel; Marcos Martin-Rincon; Paloma Martin-Sanz; José A Martina; Wim Martinet; Aitor Martinez; Ana Martinez; Jennifer Martinez; Moises Martinez Velazquez; Nuria Martinez-Lopez; Marta Martinez-Vicente; Daniel O Martins; Joilson O Martins; Waleska K Martins; Tania Martins-Marques; Emanuele Marzetti; Shashank Masaldan; Celine Masclaux-Daubresse; Douglas G Mashek; Valentina Massa; Lourdes Massieu; Glenn R Masson; Laura Masuelli; Anatoliy I Masyuk; Tetyana V Masyuk; Paola Matarrese; Ander Matheu; Satoaki Matoba; Sachiko Matsuzaki; Pamela Mattar; Alessandro Matte; Domenico Mattoscio; José L Mauriz; Mario Mauthe; Caroline Mauvezin; Emanual Maverakis; Paola Maycotte; Johanna Mayer; Gianluigi Mazzoccoli; Cristina Mazzoni; Joseph R Mazzulli; Nami McCarty; Christine McDonald; Mitchell R McGill; Sharon L McKenna; BethAnn McLaughlin; Fionn McLoughlin; Mark A McNiven; Thomas G McWilliams; Fatima Mechta-Grigoriou; Tania Catarina Medeiros; Diego L Medina; Lynn A Megeney; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Alfred J Meijer; Annemarie H Meijer; Jakob Mejlvang; Alicia Meléndez; Annette Melk; Gonen Memisoglu; Alexandrina F Mendes; Delong Meng; Fei Meng; Tian Meng; Rubem Menna-Barreto; Manoj B Menon; Carol Mercer; Anne E Mercier; Jean-Louis Mergny; Adalberto Merighi; Seth D Merkley; Giuseppe Merla; Volker Meske; Ana Cecilia Mestre; Shree Padma Metur; Christian Meyer; Hemmo Meyer; Wenyi Mi; Jeanne Mialet-Perez; Junying Miao; Lucia Micale; Yasuo Miki; Enrico Milan; Małgorzata Milczarek; Dana L Miller; Samuel I Miller; Silke Miller; Steven W Millward; Ira Milosevic; Elena A Minina; Hamed Mirzaei; Hamid Reza Mirzaei; Mehdi Mirzaei; Amit Mishra; Nandita Mishra; Paras Kumar Mishra; Maja Misirkic Marjanovic; Roberta Misasi; Amit Misra; Gabriella Misso; Claire Mitchell; Geraldine Mitou; Tetsuji Miura; Shigeki Miyamoto; Makoto Miyazaki; Mitsunori Miyazaki; Taiga Miyazaki; Keisuke Miyazawa; Noboru Mizushima; Trine H Mogensen; Baharia Mograbi; Reza Mohammadinejad; Yasir Mohamud; Abhishek Mohanty; Sipra Mohapatra; Torsten Möhlmann; Asif Mohmmed; Anna Moles; Kelle H Moley; Maurizio Molinari; Vincenzo Mollace; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Costanza Montagna; Mervyn J Monteiro; Andrea Montella; L Ruth Montes; Barbara Montico; Vinod K Mony; Giacomo Monzio Compagnoni; Michael N Moore; Mohammad A Moosavi; Ana L Mora; Marina Mora; David Morales-Alamo; Rosario Moratalla; Paula I Moreira; Elena Morelli; Sandra Moreno; Daniel Moreno-Blas; Viviana Moresi; Benjamin Morga; Alwena H Morgan; Fabrice Morin; Hideaki Morishita; Orson L Moritz; Mariko Moriyama; Yuji Moriyasu; Manuela Morleo; Eugenia Morselli; Jose F Moruno-Manchon; Jorge Moscat; Serge Mostowy; Elisa Motori; Andrea Felinto Moura; Naima Moustaid-Moussa; Maria Mrakovcic; Gabriel Muciño-Hernández; Anupam Mukherjee; Subhadip Mukhopadhyay; Jean M Mulcahy Levy; Victoriano Mulero; Sylviane Muller; Christian Münch; Ashok Munjal; Pura Munoz-Canoves; Teresa Muñoz-Galdeano; Christian Münz; Tomokazu Murakawa; Claudia Muratori; Brona M Murphy; J Patrick Murphy; Aditya Murthy; Timo T Myöhänen; Indira U Mysorekar; Jennifer Mytych; Seyed Mohammad Nabavi; Massimo Nabissi; Péter Nagy; Jihoon Nah; Aimable Nahimana; Ichiro Nakagawa; Ken Nakamura; Hitoshi Nakatogawa; Shyam S Nandi; Meera Nanjundan; Monica Nanni; Gennaro Napolitano; Roberta Nardacci; Masashi Narita; Melissa Nassif; Ilana Nathan; Manabu Natsumeda; Ryno J Naude; Christin Naumann; Olaia Naveiras; Fatemeh Navid; Steffan T Nawrocki; Taras Y Nazarko; Francesca Nazio; Florentina Negoita; Thomas Neill; Amanda L Neisch; Luca M Neri; Mihai G Netea; Patrick Neubert; Thomas P Neufeld; Dietbert Neumann; Albert Neutzner; Phillip T Newton; Paul A Ney; Ioannis P Nezis; Charlene C W Ng; Tzi Bun Ng; Hang T T Nguyen; Long T Nguyen; Hong-Min Ni; Clíona Ní Cheallaigh; Zhenhong Ni; M Celeste Nicolao; Francesco Nicoli; Manuel Nieto-Diaz; Per Nilsson; Shunbin Ning; Rituraj Niranjan; Hiroshi Nishimune; Mireia Niso-Santano; Ralph A Nixon; Annalisa Nobili; Clevio Nobrega; Takeshi Noda; Uxía Nogueira-Recalde; Trevor M Nolan; Ivan Nombela; Ivana Novak; Beatriz Novoa; Takashi Nozawa; Nobuyuki Nukina; Carmen Nussbaum-Krammer; Jesper Nylandsted; Tracey R O'Donovan; Seónadh M O'Leary; Eyleen J O'Rourke; Mary P O'Sullivan; Timothy E O'Sullivan; Salvatore Oddo; Ina Oehme; Michinaga Ogawa; Eric Ogier-Denis; Margret H Ogmundsdottir; Besim Ogretmen; Goo Taeg Oh; Seon-Hee Oh; Young J Oh; Takashi Ohama; Yohei Ohashi; Masaki Ohmuraya; Vasileios Oikonomou; Rani Ojha; Koji Okamoto; Hitoshi Okazawa; Masahide Oku; Sara Oliván; Jorge M A Oliveira; Michael Ollmann; James A Olzmann; Shakib Omari; M Bishr Omary; Gizem Önal; Martin Ondrej; Sang-Bing Ong; Sang-Ging Ong; Anna Onnis; Juan A Orellana; Sara Orellana-Muñoz; Maria Del Mar Ortega-Villaizan; Xilma R Ortiz-Gonzalez; Elena Ortona; Heinz D Osiewacz; Abdel-Hamid K Osman; Rosario Osta; Marisa S Otegui; Kinya Otsu; Christiane Ott; Luisa Ottobrini; Jing-Hsiung James Ou; Tiago F Outeiro; Inger Oynebraten; Melek Ozturk; Gilles Pagès; Susanta Pahari; Marta Pajares; Utpal B Pajvani; Rituraj Pal; Simona Paladino; Nicolas Pallet; Michela Palmieri; Giuseppe Palmisano; Camilla Palumbo; Francesco Pampaloni; Lifeng Pan; Qingjun Pan; Wenliang Pan; Xin Pan; Ganna Panasyuk; Rahul Pandey; Udai B Pandey; Vrajesh Pandya; Francesco Paneni; Shirley Y Pang; Elisa Panzarini; Daniela L Papademetrio; Elena Papaleo; Daniel Papinski; Diana Papp; Eun Chan Park; Hwan Tae Park; Ji-Man Park; Jong-In Park; Joon Tae Park; Junsoo Park; Sang Chul Park; Sang-Youel Park; Abraham H Parola; Jan B Parys; Adrien Pasquier; Benoit Pasquier; João F Passos; Nunzia Pastore; Hemal H Patel; Daniel Patschan; Sophie Pattingre; Gustavo Pedraza-Alva; Jose Pedraza-Chaverri; Zully Pedrozo; Gang Pei; Jianming Pei; Hadas Peled-Zehavi; Joaquín M Pellegrini; Joffrey Pelletier; Miguel A Peñalva; Di Peng; Ying Peng; Fabio Penna; Maria Pennuto; Francesca Pentimalli; Cláudia Mf Pereira; Gustavo J S Pereira; Lilian C Pereira; Luis Pereira de Almeida; Nirma D Perera; Ángel Pérez-Lara; Ana B Perez-Oliva; María Esther Pérez-Pérez; Palsamy Periyasamy; Andras Perl; Cristiana Perrotta; Ida Perrotta; Richard G Pestell; Morten Petersen; Irina Petrache; Goran Petrovski; Thorsten Pfirrmann; Astrid S Pfister; Jennifer A Philips; Huifeng Pi; Anna Picca; Alicia M Pickrell; Sandy Picot; Giovanna M Pierantoni; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Karolina Pierzynowska; Federico Pietrocola; Miroslawa Pietruczuk; Claudio Pignata; Felipe X Pimentel-Muiños; Mario Pinar; Roberta O Pinheiro; Ronit Pinkas-Kramarski; Paolo Pinton; Karolina Pircs; Sujan Piya; Paola Pizzo; Theo S Plantinga; Harald W Platta; Ainhoa Plaza-Zabala; Markus Plomann; Egor Y Plotnikov; Helene Plun-Favreau; Ryszard Pluta; Roger Pocock; Stefanie Pöggeler; Christian Pohl; Marc Poirot; Angelo Poletti; Marisa Ponpuak; Hana Popelka; Blagovesta Popova; Helena Porta; Soledad Porte Alcon; Eliana Portilla-Fernandez; Martin Post; Malia B Potts; Joanna Poulton; Ted Powers; Veena Prahlad; Tomasz K Prajsnar; Domenico Praticò; Rosaria Prencipe; Muriel Priault; Tassula Proikas-Cezanne; Vasilis J Promponas; Christopher G Proud; Rosa Puertollano; Luigi Puglielli; Thomas Pulinilkunnil; Deepika Puri; Rajat Puri; Julien Puyal; Xiaopeng Qi; Yongmei Qi; Wenbin Qian; Lei Qiang; Yu Qiu; Joe Quadrilatero; Jorge Quarleri; Nina Raben; Hannah Rabinowich; Debora Ragona; Michael J Ragusa; Nader Rahimi; Marveh Rahmati; Valeria Raia; Nuno Raimundo; Namakkal-Soorappan Rajasekaran; Sriganesh Ramachandra Rao; Abdelhaq Rami; Ignacio Ramírez-Pardo; David B Ramsden; Felix Randow; Pundi N Rangarajan; Danilo Ranieri; Hai Rao; Lang Rao; Rekha Rao; Sumit Rathore; J Arjuna Ratnayaka; Edward A Ratovitski; Palaniyandi Ravanan; Gloria Ravegnini; Swapan K Ray; Babak Razani; Vito Rebecca; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; David Reigada; Jan H Reiling; Theo Rein; Siegfried Reipert; Rokeya Sultana Rekha; Hongmei Ren; Jun Ren; Weichao Ren; Tristan Renault; Giorgia Renga; Karen Reue; Kim Rewitz; Bruna Ribeiro de Andrade Ramos; S Amer Riazuddin; Teresa M Ribeiro-Rodrigues; Jean-Ehrland Ricci; Romeo Ricci; Victoria Riccio; Des R Richardson; Yasuko Rikihisa; Makarand V Risbud; Ruth M Risueño; Konstantinos Ritis; Salvatore Rizza; Rosario Rizzuto; Helen C Roberts; Luke D Roberts; Katherine J Robinson; Maria Carmela Roccheri; Stephane Rocchi; George G Rodney; Tiago Rodrigues; Vagner Ramon Rodrigues Silva; Amaia Rodriguez; Ruth Rodriguez-Barrueco; Nieves Rodriguez-Henche; Humberto Rodriguez-Rocha; Jeroen Roelofs; Robert S Rogers; Vladimir V Rogov; Ana I Rojo; Krzysztof Rolka; Vanina Romanello; Luigina Romani; Alessandra Romano; Patricia S Romano; David Romeo-Guitart; Luis C Romero; Montserrat Romero; Joseph C Roney; Christopher Rongo; Sante Roperto; Mathias T Rosenfeldt; Philip Rosenstiel; Anne G Rosenwald; Kevin A Roth; Lynn Roth; Steven Roth; Kasper M A Rouschop; Benoit D Roussel; Sophie Roux; Patrizia Rovere-Querini; Ajit Roy; Aurore Rozieres; Diego Ruano; David C Rubinsztein; Maria P Rubtsova; Klaus Ruckdeschel; Christoph Ruckenstuhl; Emil Rudolf; Rüdiger Rudolf; Alessandra Ruggieri; Avnika Ashok Ruparelia; Paola Rusmini; Ryan R Russell; Gian Luigi Russo; Maria Russo; Rossella Russo; Oxana O Ryabaya; Kevin M Ryan; Kwon-Yul Ryu; Maria Sabater-Arcis; Ulka Sachdev; Michael Sacher; Carsten Sachse; Abhishek Sadhu; Junichi Sadoshima; Nathaniel Safren; Paul Saftig; Antonia P Sagona; Gaurav Sahay; Amirhossein Sahebkar; Mustafa Sahin; Ozgur Sahin; Sumit Sahni; Nayuta Saito; Shigeru Saito; Tsunenori Saito; Ryohei Sakai; Yasuyoshi Sakai; Jun-Ichi Sakamaki; Kalle Saksela; Gloria Salazar; Anna Salazar-Degracia; Ghasem H Salekdeh; Ashok K Saluja; Belém Sampaio-Marques; Maria Cecilia Sanchez; Jose A Sanchez-Alcazar; Victoria Sanchez-Vera; Vanessa Sancho-Shimizu; J Thomas Sanderson; Marco Sandri; Stefano Santaguida; Laura Santambrogio; Magda M Santana; Giorgio Santoni; Alberto Sanz; Pascual Sanz; Shweta Saran; Marco Sardiello; Timothy J Sargeant; Apurva Sarin; Chinmoy Sarkar; Sovan Sarkar; Maria-Rosa Sarrias; Surajit Sarkar; Dipanka Tanu Sarmah; Jaakko Sarparanta; Aishwarya Sathyanarayan; Ranganayaki Sathyanarayanan; K Matthew Scaglione; Francesca Scatozza; Liliana Schaefer; Zachary T Schafer; Ulrich E Schaible; Anthony H V Schapira; Michael Scharl; Hermann M Schatzl; Catherine H Schein; Wiep Scheper; David Scheuring; Maria Vittoria Schiaffino; Monica Schiappacassi; Rainer Schindl; Uwe Schlattner; Oliver Schmidt; Roland Schmitt; Stephen D Schmidt; Ingo Schmitz; Eran Schmukler; Anja Schneider; Bianca E Schneider; Romana Schober; Alejandra C Schoijet; Micah B Schott; Michael Schramm; Bernd Schröder; Kai Schuh; Christoph Schüller; Ryan J Schulze; Lea Schürmanns; Jens C Schwamborn; Melanie Schwarten; Filippo Scialo; Sebastiano Sciarretta; Melanie J Scott; Kathleen W Scotto; A Ivana Scovassi; Andrea Scrima; Aurora Scrivo; David Sebastian; Salwa Sebti; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Iban Seiliez; Ekihiro Seki; Scott B Selleck; Frank W Sellke; Joshua T Selsby; Michael Sendtner; Serif Senturk; Elena Seranova; Consolato Sergi; Ruth Serra-Moreno; Hiromi Sesaki; Carmine Settembre; Subba Rao Gangi Setty; Gianluca Sgarbi; Ou Sha; John J Shacka; Javeed A Shah; Dantong Shang; Changshun Shao; Feng Shao; Soroush Sharbati; Lisa M Sharkey; Dipali Sharma; Gaurav Sharma; Kulbhushan Sharma; Pawan Sharma; Surendra Sharma; Han-Ming Shen; Hongtao Shen; Jiangang Shen; Ming Shen; Weili Shen; Zheni Shen; Rui Sheng; Zhi Sheng; Zu-Hang Sheng; Jianjian Shi; Xiaobing Shi; Ying-Hong Shi; Kahori Shiba-Fukushima; Jeng-Jer Shieh; Yohta Shimada; Shigeomi Shimizu; Makoto Shimozawa; Takahiro Shintani; Christopher J Shoemaker; Shahla Shojaei; Ikuo Shoji; Bhupendra V Shravage; Viji Shridhar; Chih-Wen Shu; Hong-Bing Shu; Ke Shui; Arvind K Shukla; Timothy E Shutt; Valentina Sica; Aleem Siddiqui; Amanda Sierra; Virginia Sierra-Torre; Santiago Signorelli; Payel Sil; Bruno J de Andrade Silva; Johnatas D Silva; Eduardo Silva-Pavez; Sandrine Silvente-Poirot; Rachel E Simmonds; Anna Katharina Simon; Hans-Uwe Simon; Matias Simons; Anurag Singh; Lalit P Singh; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Sudha B Singh; Sunaina Singh; Surinder Pal Singh; Debasish Sinha; Rohit Anthony Sinha; Sangita Sinha; Agnieszka Sirko; Kapil Sirohi; Efthimios L Sivridis; Panagiotis Skendros; Aleksandra Skirycz; Iva Slaninová; Soraya S Smaili; Andrei Smertenko; Matthew D Smith; Stefaan J Soenen; Eun Jung Sohn; Sophia P M Sok; Giancarlo Solaini; Thierry Soldati; Scott A Soleimanpour; Rosa M Soler; Alexei Solovchenko; Jason A Somarelli; Avinash Sonawane; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Kunhua Song; Zhiyin Song; Leandro R Soria; Maurizio Sorice; Alexander A Soukas; Sandra-Fausia Soukup; Diana Sousa; Nadia Sousa; Paul A Spagnuolo; Stephen A Spector; M M Srinivas Bharath; Daret St Clair; Venturina Stagni; Leopoldo Staiano; Clint A Stalnecker; Metodi V Stankov; Peter B Stathopulos; Katja Stefan; Sven Marcel Stefan; Leonidas Stefanis; Joan S Steffan; Alexander Steinkasserer; Harald Stenmark; Jared Sterneckert; Craig Stevens; Veronika Stoka; Stephan Storch; Björn Stork; Flavie Strappazzon; Anne Marie Strohecker; Dwayne G Stupack; Huanxing Su; Ling-Yan Su; Longxiang Su; Ana M Suarez-Fontes; Carlos S Subauste; Selvakumar Subbian; Paula V Subirada; Ganapasam Sudhandiran; Carolyn M Sue; Xinbing Sui; Corey Summers; Guangchao Sun; Jun Sun; Kang Sun; Meng-Xiang Sun; Qiming Sun; Yi Sun; Zhongjie Sun; Karen K S Sunahara; Eva Sundberg; Katalin Susztak; Peter Sutovsky; Hidekazu Suzuki; Gary Sweeney; J David Symons; Stephen Cho Wing Sze; Nathaniel J Szewczyk; Anna Tabęcka-Łonczynska; Claudio Tabolacci; Frank Tacke; Heinrich Taegtmeyer; Marco Tafani; Mitsuo Tagaya; Haoran Tai; Stephen W G Tait; Yoshinori Takahashi; Szabolcs Takats; Priti Talwar; Chit Tam; Shing Yau Tam; Davide Tampellini; Atsushi Tamura; Chong Teik Tan; Eng-King Tan; Ya-Qin Tan; Masaki Tanaka; Motomasa Tanaka; Daolin Tang; Jingfeng Tang; Tie-Shan Tang; Isei Tanida; Zhipeng Tao; Mohammed Taouis; Lars Tatenhorst; Nektarios Tavernarakis; Allen Taylor; Gregory A Taylor; Joan M Taylor; Elena Tchetina; Andrew R Tee; Irmgard Tegeder; David Teis; Natercia Teixeira; Fatima Teixeira-Clerc; Kumsal A Tekirdag; Tewin Tencomnao; Sandra Tenreiro; Alexei V Tepikin; Pilar S Testillano; Gianluca Tettamanti; Pierre-Louis Tharaux; Kathrin Thedieck; Arvind A Thekkinghat; Stefano Thellung; Josephine W Thinwa; V P Thirumalaikumar; Sufi Mary Thomas; Paul G Thomes; Andrew Thorburn; Lipi Thukral; Thomas Thum; Michael Thumm; Ling Tian; Ales Tichy; Andreas Till; Vincent Timmerman; Vladimir I Titorenko; Sokol V Todi; Krassimira Todorova; Janne M Toivonen; Luana Tomaipitinca; Dhanendra Tomar; Cristina Tomas-Zapico; Sergej Tomić; Benjamin Chun-Kit Tong; Chao Tong; Xin Tong; Sharon A Tooze; Maria L Torgersen; Satoru Torii; Liliana Torres-López; Alicia Torriglia; Christina G Towers; Roberto Towns; Shinya Toyokuni; Vladimir Trajkovic; Donatella Tramontano; Quynh-Giao Tran; Leonardo H Travassos; Charles B Trelford; Shirley Tremel; Ioannis P Trougakos; Betty P Tsao; Mario P Tschan; Hung-Fat Tse; Tak Fu Tse; Hitoshi Tsugawa; Andrey S Tsvetkov; David A Tumbarello; Yasin Tumtas; María J Tuñón; Sandra Turcotte; Boris Turk; Vito Turk; Bradley J Turner; Richard I Tuxworth; Jessica K Tyler; Elena V Tyutereva; Yasuo Uchiyama; Aslihan Ugun-Klusek; Holm H Uhlig; Marzena Ułamek-Kozioł; Ilya V Ulasov; Midori Umekawa; Christian Ungermann; Rei Unno; Sylvie Urbe; Elisabet Uribe-Carretero; Suayib Üstün; Vladimir N Uversky; Thomas Vaccari; Maria I Vaccaro; Björn F Vahsen; Helin Vakifahmetoglu-Norberg; Rut Valdor; Maria J Valente; Ayelén Valko; Richard B Vallee; Angela M Valverde; Greet Van den Berghe; Stijn van der Veen; Luc Van Kaer; Jorg van Loosdregt; Sjoerd J L van Wijk; Wim Vandenberghe; Ilse Vanhorebeek; Marcos A Vannier-Santos; Nicola Vannini; M Cristina Vanrell; Chiara Vantaggiato; Gabriele Varano; Isabel Varela-Nieto; Máté Varga; M Helena Vasconcelos; Somya Vats; Demetrios G Vavvas; Ignacio Vega-Naredo; Silvia Vega-Rubin-de-Celis; Guillermo Velasco; Ariadna P Velázquez; Tibor Vellai; Edo Vellenga; Francesca Velotti; Mireille Verdier; Panayotis Verginis; Isabelle Vergne; Paul Verkade; Manish Verma; Patrik Verstreken; Tim Vervliet; Jörg Vervoorts; Alexandre T Vessoni; Victor M Victor; Michel Vidal; Chiara Vidoni; Otilia V Vieira; Richard D Vierstra; Sonia Viganó; Helena Vihinen; Vinoy Vijayan; Miquel Vila; Marçal Vilar; José M Villalba; Antonio Villalobo; Beatriz Villarejo-Zori; Francesc Villarroya; Joan Villarroya; Olivier Vincent; Cecile Vindis; Christophe Viret; Maria Teresa Viscomi; Dora Visnjic; Ilio Vitale; David J Vocadlo; Olga V Voitsekhovskaja; Cinzia Volonté; Mattia Volta; Marta Vomero; Clarissa Von Haefen; Marc A Vooijs; Wolfgang Voos; Ljubica Vucicevic; Richard Wade-Martins; Satoshi Waguri; Kenrick A Waite; Shuji Wakatsuki; David W Walker; Mark J Walker; Simon A Walker; Jochen Walter; Francisco G Wandosell; Bo Wang; Chao-Yung Wang; Chen Wang; Chenran Wang; Chenwei Wang; Cun-Yu Wang; Dong Wang; Fangyang Wang; Feng Wang; Fengming Wang; Guansong Wang; Han Wang; Hao Wang; Hexiang Wang; Hong-Gang Wang; Jianrong Wang; Jigang Wang; Jiou Wang; Jundong Wang; Kui Wang; Lianrong Wang; Liming Wang; Maggie Haitian Wang; Meiqing Wang; Nanbu Wang; Pengwei Wang; Peipei Wang; Ping Wang; Ping Wang; Qing Jun Wang; Qing Wang; Qing Kenneth Wang; Qiong A Wang; Wen-Tao Wang; Wuyang Wang; Xinnan Wang; Xuejun Wang; Yan Wang; Yanchang Wang; Yanzhuang Wang; Yen-Yun Wang; Yihua Wang; Yipeng Wang; Yu Wang; Yuqi Wang; Zhe Wang; Zhenyu Wang; Zhouguang Wang; Gary Warnes; Verena Warnsmann; Hirotaka Watada; Eizo Watanabe; Maxinne Watchon; Anna Wawrzyńska; Timothy E Weaver; Grzegorz Wegrzyn; Ann M Wehman; Huafeng Wei; Lei Wei; Taotao Wei; Yongjie Wei; Oliver H Weiergräber; Conrad C Weihl; Günther Weindl; Ralf Weiskirchen; Alan Wells; Runxia H Wen; Xin Wen; Antonia Werner; Beatrice Weykopf; Sally P Wheatley; J Lindsay Whitton; Alexander J Whitworth; Katarzyna Wiktorska; Manon E Wildenberg; Tom Wileman; Simon Wilkinson; Dieter Willbold; Brett Williams; Robin S B Williams; Roger L Williams; Peter R Williamson; Richard A Wilson; Beate Winner; Nathaniel J Winsor; Steven S Witkin; Harald Wodrich; Ute Woehlbier; Thomas Wollert; Esther Wong; Jack Ho Wong; Richard W Wong; Vincent Kam Wai Wong; W Wei-Lynn Wong; An-Guo Wu; Chengbiao Wu; Jian Wu; Junfang Wu; Kenneth K Wu; Min Wu; Shan-Ying Wu; Shengzhou Wu; Shu-Yan Wu; Shufang Wu; William K K Wu; Xiaohong Wu; Xiaoqing Wu; Yao-Wen Wu; Yihua Wu; Ramnik J Xavier; Hongguang Xia; Lixin Xia; Zhengyuan Xia; Ge Xiang; Jin Xiang; Mingliang Xiang; Wei Xiang; Bin Xiao; Guozhi Xiao; Hengyi Xiao; Hong-Tao Xiao; Jian Xiao; Lan Xiao; Shi Xiao; Yin Xiao; Baoming Xie; Chuan-Ming Xie; Min Xie; Yuxiang Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Congfeng Xu; En Xu; Haoxing Xu; Jing Xu; JinRong Xu; Liang Xu; Wen Wen Xu; Xiulong Xu; Yu Xue; Sokhna M S Yakhine-Diop; Masamitsu Yamaguchi; Osamu Yamaguchi; Ai Yamamoto; Shunhei Yamashina; Shengmin Yan; Shian-Jang Yan; Zhen Yan; Yasuo Yanagi; Chuanbin Yang; Dun-Sheng Yang; Huan Yang; Huang-Tian Yang; Hui Yang; Jin-Ming Yang; Jing Yang; Jingyu Yang; Ling Yang; Liu Yang; Ming Yang; Pei-Ming Yang; Qian Yang; Seungwon Yang; Shu Yang; Shun-Fa Yang; Wannian Yang; Wei Yuan Yang; Xiaoyong Yang; Xuesong Yang; Yi Yang; Ying Yang; Honghong Yao; Shenggen Yao; Xiaoqiang Yao; Yong-Gang Yao; Yong-Ming Yao; Takahiro Yasui; Meysam Yazdankhah; Paul M Yen; Cong Yi; Xiao-Ming Yin; Yanhai Yin; Zhangyuan Yin; Ziyi Yin; Meidan Ying; Zheng Ying; Calvin K Yip; Stephanie Pei Tung Yiu; Young H Yoo; Kiyotsugu Yoshida; Saori R Yoshii; Tamotsu Yoshimori; Bahman Yousefi; Boxuan Yu; Haiyang Yu; Jun Yu; Jun Yu; Li Yu; Ming-Lung Yu; Seong-Woon Yu; Victor C Yu; W Haung Yu; Zhengping Yu; Zhou Yu; Junying Yuan; Ling-Qing Yuan; Shilin Yuan; Shyng-Shiou F Yuan; Yanggang Yuan; Zengqiang Yuan; Jianbo Yue; Zhenyu Yue; Jeanho Yun; Raymond L Yung; David N Zacks; Gabriele Zaffagnini; Vanessa O Zambelli; Isabella Zanella; Qun S Zang; Sara Zanivan; Silvia Zappavigna; Pilar Zaragoza; Konstantinos S Zarbalis; Amir Zarebkohan; Amira Zarrouk; Scott O Zeitlin; Jialiu Zeng; Ju-Deng Zeng; Eva Žerovnik; Lixuan Zhan; Bin Zhang; Donna D Zhang; Hanlin Zhang; Hong Zhang; Hong Zhang; Honghe Zhang; Huafeng Zhang; Huaye Zhang; Hui Zhang; Hui-Ling Zhang; Jianbin Zhang; Jianhua Zhang; Jing-Pu Zhang; Kalin Y B Zhang; Leshuai W Zhang; Lin Zhang; Lisheng Zhang; Lu Zhang; Luoying Zhang; Menghuan Zhang; Peng Zhang; Sheng Zhang; Wei Zhang; Xiangnan Zhang; Xiao-Wei Zhang; Xiaolei Zhang; Xiaoyan Zhang; Xin Zhang; Xinxin Zhang; Xu Dong Zhang; Yang Zhang; Yanjin Zhang; Yi Zhang; Ying-Dong Zhang; Yingmei Zhang; Yuan-Yuan Zhang; Yuchen Zhang; Zhe Zhang; Zhengguang Zhang; Zhibing Zhang; Zhihai Zhang; Zhiyong Zhang; Zili Zhang; Haobin Zhao; Lei Zhao; Shuang Zhao; Tongbiao Zhao; Xiao-Fan Zhao; Ying Zhao; Yongchao Zhao; Yongliang Zhao; Yuting Zhao; Guoping Zheng; Kai Zheng; Ling Zheng; Shizhong Zheng; Xi-Long Zheng; Yi Zheng; Zu-Guo Zheng; Boris Zhivotovsky; Qing Zhong; Ao Zhou; Ben Zhou; Cefan Zhou; Gang Zhou; Hao Zhou; Hong Zhou; Hongbo Zhou; Jie Zhou; Jing Zhou; Jing Zhou; Jiyong Zhou; Kailiang Zhou; Rongjia Zhou; Xu-Jie Zhou; Yanshuang Zhou; Yinghong Zhou; Yubin Zhou; Zheng-Yu Zhou; Zhou Zhou; Binglin Zhu; Changlian Zhu; Guo-Qing Zhu; Haining Zhu; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Yanping Zhu; Yushan Zhu; Haixia Zhuang; Xiaohong Zhuang; Katarzyna Zientara-Rytter; Christine M Zimmermann; Elena Ziviani; Teresa Zoladek; Wei-Xing Zong; Dmitry B Zorov; Antonio Zorzano; Weiping Zou; Zhen Zou; Zhengzhi Zou; Steven Zuryn; Werner Zwerschke; Beate Brand-Saberi; X Charlie Dong; Chandra Shekar Kenchappa; Zuguo Li; Yong Lin; Shigeru Oshima; Yueguang Rong; Judith C Sluimer; Christina L Stallings; Chun-Kit Tong Journal: Autophagy Date: 2021-02-08 Impact factor: 13.391
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