Yongwang Zhong

Heat-shock protein 90α1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos

Heat-shock protein 90α (Hsp90α) is a member of the molecular chaperone family involved in protein folding and assembly. The role of Hsp90α in the developmental process, however, remains unclear. Here we report that zebrafish contains two Hsp90α genes, Hsp90α1, and Hsp90α2. Hsp90α1 is specifically expressed in developing somites and skeletal muscles of zebrafish embryos. We have demonstrated that Hsp90α1 is essential for myofibril organization in skeletal muscles of zebrafish embryos. Knockdown of Hsp90α1 resulted in paralyzed zebrafish embryos with poorly organized myofibrils in skeletal muscles. In contrast, knockdown of Hsp90α2 had no effect on muscle contraction and myofibril organization. The filament defects could be rescued in a cell autonomous manner by an ectopic expression of Hsp90α1. Biochemical analyses revealed that knockdown of Hsp90α1 resulted in significant myosin degradation and up-regulation of unc-45b gene expression. These results indicate that Hsp90α1 plays an important role in muscle development, likely through facilitating myosin folding and assembly into organized myofibril filaments.

Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP

Huntingtons disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3δ from association with gp78 and p97/VCP, which is accompanied by decreases in CD3δ ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3δ and misfolded Z variant of α-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.

Smyd1b is required for skeletal and cardiac muscle function in zebrafish

Myofibrillogenesis is critical for muscle cell differentiation and contraction. This study shows that Smyd1b plays a key role in myofibrillogenesis in muscle cells. Knockdown of smyd1b results in up-regulation of hsp90α1 and unc45b gene expression, increased myosin degradation, and disruption of sarcomere organization in zebrafish embryos.

Identification of a Naturally Occurring Recombinant Isolate of Sugarcane Mosaic Virus Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 ′-terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5′-untranslated region (UTR) and a 255 nt 3′-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.

Rice black‐streaked dwarf virus minor core protein P8 is a nuclear dimeric protein and represses transcription in tobacco protoplasts

Virus‐encoding nuclear transcriptional regulators play important roles in the viral life cycle. Most of these proteins exhibit intrinsic transcriptional activation or repression activity, and are involved in the regulation of the expression of virus genome itself or important cellular genes to facilitate viral replication and inhibit antiviral responses. Here, we report that the minor core protein P8 of Rice black‐streaked dwarf virus, a dsRNA virus infecting host plants and insects, is targeted to the nucleus of insect and plant cells via its N‐terminal 1–40 amino acids and possesses potent active transcriptional repression activity in Bright Yellow‐2 tobacco suspension cells. Moreover, P8, like many transcriptional regulatory proteins, is capable of forming homo‐dimers within insect cells and in vitro. All these data suggest that P8 is likely to enter the nucleus of host cell and play an important role as a negative transcriptional regulator of host gene expression during the process of virus–host interaction.

Live Cell Imaging Of Protein Dislocation From The Endoplasmic Reticulum

Background: Dislocation of proteins from the endoplasmic reticulum to the cytosol is an essential cellular process. Results: Dislocation of engineered proteins leads to reconstitution of split GFP fragments in living cells. Conclusion: A GFP reporter has been developed for studying dislocation in living cells. Significance: The strategy is broadly applicable to the study of transmembrane transport of proteins and likely also of viruses and toxins. Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.

Importin β Interacts with the Endoplasmic Reticulum-associated Degradation Machinery and Promotes Ubiquitination and Degradation of Mutant α1-Antitrypsin

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.

ER stress response during the differentiation of H9 cells induced by retinoic acid.

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2α was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2α was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells, but its downregulation was much slower in HEK293 cells. Additionally, two ER-resident E3 ubiquitin ligases, gp78 and Hrd1, were both upregulated in H9 cells following 5 days of exposure to RA. Moreover, the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells, and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29 days, GRP78/Bip, XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA.

Importin β interacts with the ER-associated degradation machinery and promotes ubiquitination and degradation of mutant α1-antitrypsin

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.