Role In Protein Quality Control Research Articles

Endoplasmic reticulum stress and its role in various neurodegenerative diseases

The Endoplasmic reticulum (ER), a critical cellular organelle, maintains cellular homeostasis by regulating calcium levels and orchestrating essential functions such as protein synthesis, folding, and lipid production. A pivotal aspect of ER function is its role in protein quality control. When misfolded proteins accumulate within the ER due to factors like protein folding chaperone dysfunction, toxicity, oxidative stress, or inflammation, it triggers the Unfolded protein response (UPR). The UPR involves the activation of chaperones like calnexin, calreticulin, glucose-regulating protein 78 (GRP78), and Glucose-regulating protein 94 (GRP94), along with oxidoreductases like protein disulphide isomerases (PDIs). Cells employ the Endoplasmic reticulum-associated degradation (ERAD) mechanism to counteract protein misfolding. ERAD disruption causes the detachment of GRP78 from transmembrane proteins, initiating a cascade involving Inositol-requiring kinase/endoribonuclease 1 (IRE1), Activating transcription factor 6 (ATF6), and Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathways. The accumulation and deposition of misfolded proteins within the cell are hallmarks of numerous neurodegenerative diseases. These aberrant proteins disrupt normal neuronal signalling and contribute to impaired cellular homeostasis, including oxidative stress and compromised protein degradation pathways. In essence, ER stress is defined as the cellular response to the accumulation of misfolded proteins in the endoplasmic reticulum, encompassing a series of signalling pathways and molecular events that aim to restore cellular homeostasis. This comprehensive review explores ER stress and its profound implications for the pathogenesis and progression of neurodegenerative diseases.

Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans.

Candida albicans is the most prevalent human fungal pathogen. In immunocompromised individuals, C. albicans can cause serious systemic disease, and patients infected with drug-resistant isolates have few treatment options. The ubiquitin-proteasome system has not been thoroughly characterized in C. albicans. Research from other organisms has shown ubiquitination is important for protein quality control and regulated protein degradation at the endoplasmic reticulum (ER) via ER-associated protein degradation (ERAD). Here we perform the first characterization, to our knowledge, of ERAD in a human fungal pathogen. We generated functional knockouts of C. albicans genes encoding three proteins predicted to play roles in ERAD, the ubiquitin ligases Hrd1 and Doa10 and the ubiquitin-conjugating enzyme Ubc7. We assessed the fitness of each mutant in the presence of proteotoxic stress, and we used quantitative tandem mass tag mass spectrometry to characterize proteomic alterations in yeast lacking each gene. Consistent with a role in protein quality control, yeast lacking proteins thought to contribute to ERAD displayed hypersensitivity to proteotoxic stress. Furthermore, each mutant displayed distinct proteomic profiles, revealing potential physiological ERAD substrates, co-factors, and compensatory stress response factors. Among candidate ERAD substrates are enzymes contributing to ergosterol synthesis, a known therapeutic vulnerability of C. albicans. Together, our results provide the first description of ERAD function in C. albicans, and, to our knowledge, any pathogenic fungus.

Syntaxin 5-dependent phosphorylation of the small heat shock protein Hsp42 and its role in protein quality control.

The small heat shock protein Hsp42 and the t-SNARE protein Sed5 have central roles in the sequestration of misfolded proteins into insoluble protein deposits in the yeast Saccharomyces cerevisiae. However, whether these proteins/processes interact in protein quality control (PQC) is not known. Here, we show that Sed5 and anterograde trafficking modulate phosphorylation of Hsp42 partially via the MAPK kinase Hog1. Such phosphorylation, specifically at residue S215, abrogated the co-localization of Hsp42 with the Hsp104 disaggregase, aggregate clearance, chaperone activity, and sequestration of aggregates to IPOD and mitochondria. Furthermore, we found that Hsp42 is hyperphosphorylated in old cells leading to a drastic failure in disaggregation. Old cells also displayed a retarded anterograde trafficking, which, together with slow aggregate clearance and hyperphosphorylation of Hsp42, could be counteracted by Sed5 overproduction. We hypothesize that the breakdown of proper PQC during yeast aging may, in part, be due to a retarded anterograde trafficking leading to hyperphosphorylation of Hsp42.

Transcriptome Analysis Reveals Potential Mechanism in Storage Protein Trafficking within Developing Grains of Common Wheat

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which defines the viscoelastic properties of wheat dough. The synthesis of these storage proteins is controlled by the endoplasmic reticulum (ER) and is directed into the vacuole via the Golgi apparatus. In the present study, transcriptome analysis was used to explore the potential mechanism within critical stages of grain development of wheat cultivar "Shaannong 33" and its sister line used as the control (CK). Samples were collected at 10 DPA (days after anthesis), 14 DPA, 20 DPA, and 30 DPA for transcriptomic analysis. The comparative transcriptome analysis identified that a total of 18,875 genes were differentially expressed genes (DEGs) between grains of four groups "T10 vs. CK10, T14 vs. CK14, T20 vs. CK20, and T30 vs. CK30", including 2824 up-regulated and 5423 down-regulated genes in T30 vs. CK30. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment highlighted the maximum number of genes regulating protein processing in the endoplasmic reticulum (ER) during grain enlargement stages (10-20 DPA). In addition, KEGG database analysis reported 1362 and 788 DEGs involved in translation, ribosomal structure, biogenesis, flavonoid biosynthesis pathway and intracellular trafficking, secretion, and vesicular transport through protein processing within ER pathway (ko04141). Notably, consistent with the higher expression of intercellular storage protein trafficking genes at the initial 10 DPA, there was relatively low expression at later stages. Expression levels of nine randomly selected genes were verified by qRT-PCR, which were consistent with the transcriptome data. These data suggested that the initial stages of "cell division" played a significant role in protein quality control within the ER, thus maintaining the protein quality characteristics at grain maturity. Furthermore, our data suggested that the protein synthesis, folding, and trafficking pathways directed by a different number of genes during the grain enlargement stage contributed to the observed high-quality characteristics of gluten protein in Shaannong 33 (Triticum aestivum L.).

Genetic deletion of hspa8 leads to selective tissue malformations in zebrafish embryonic development.

The heat shock cognate 71 kDa protein HSPA8 (also known as HSC70), a constitutively expressed cognate member of the heat shock protein 70 family, plays an essential role in protein quality control and cell homeostasis maintenance. HSPA8 has been implicated in many diseases, including cancers and neurodegenerative diseases. Owing to massive cell death after knockdown of HSPA8 and nonviable Hspa8 knockout mice, the physiological role of HSPA8 in vertebrates and its underlying mechanisms of action have not yet been elucidated. To address this issue, we used CRISPR/Cas9 technology and genetically deleted hspa8 in zebrafish embryos. Genetic deletion of hspa8 resulted in malformations of the pharyngeal arches, pectoral fins, head and eyes at the later stages. We next focused on pharyngeal arch deficiency and found that pharyngeal arches in hspa8 mutant embryos exhibited induction of endoplasmic reticulum stress and activation of the unfolded protein response via the Perk/p-eIF2α/Atf4 signaling cascade. Inhibition of Perk/p-eIF2α/Atf4 signaling rescued the developmental deficiency of pharyngeal arches caused by depletion of Hspa8. Taken together, our results provide novel insights into the tissue-specific roles of Hspa8 in the regulation of vertebrate embryonic development.

Activation of the Soluble Guanylate Cyclase Increases 26S Proteasome Activities and Protects against Proteotoxicity in Cardiomyocytes

BackgroundThe ubiquitin‐proteasome system (UPS) mediates the selective degradation of unwanted and unneeded proteins in the cell, playing crucial roles in protein quality control and diverse other cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of heart diseases. Our laboratory has previously discovered that activation of the cGMP‐dependent protein kinase (PKG) primes the proteasome. Here we test the hypothesis that by increasing cGMP and PKG activity, soluble guanylate cyclase (sGC) activation activates the proteasome and thereby protects against proteotoxicity in cardiomyocytes.Methods and ResultsCompared with vehicle control (DMSO), treatment with an sGC activator (cinaciguat) in cultured neonatal rat cardiomyocytes (NRCMs) led to increases in the levels of Ser239‐phosphorylated vasodilator‐stimulated phosphoprotein (VASP) (2.17±0.07 vs. 1.0±0.20, p<0.0001), indicative of PKG activation. We then used adenoviral vectors expressing degron CL1‐fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) as a reporter system for UPS performance. Compared with DMSO, cinaciguat treatment reduced the GFPu/RFP ratio (0.59±0.06 vs. 1.0±0.18, p=0.0135) as revealed by western blotting, and shortened GFPu half‐life in cultured NRCMs as revealed by cycloheximide chase assays. Cinaciguat treatment decreased Thr41/Ser45‐phosphorylated β‐catenin, an endogenous substrate of the UPS (0.60±0.12 vs. 1.0±0.08, p=0.0014). These data demonstrate that sGC activation enhances UPS performance. Moreover, in‐gel proteasome activity assays followed by western blot analyses reveal that cinaciguat treatment increased the chymotrypsin like activities of 26S proteasomes (1.52±0.17 vs. 1.0±0.09, p=0.0019) without changing 26S proteasome assembly, showing that sGC activation enhances UPS performance via activating 26S proteasomes. We next used NRCMs with adenovirus‐mediated overexpression of a human‐disease‐linked misfolded protein CryABR120Gas a cell model of cardiac proteotoxicity. CryABR120G overexpression‐induced increases in LDH leakage and PKCδ cleavage (both indicative of cell injury/death) were significantly attenuated by cinaciguat treatment (LDH in media: 0.71±0.20 vs.1.44±0.26, p<0.0001); further MTT assays consistently showed cinaciguat promoted NRCMs viability (1.32±0.11 vs. 1.02±0.05, p<0.0001).ConclusionsActivation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates UPS functioning, and thereby protects against proteotoxicity in cardiomyocytes.

The Modulatory Role of sti-1 in Methylmercury-Induced Toxicity in Caenorhabditis elegans.

Human exposure to the neurotoxin methylmercury (MeHg) poses a significant health risk to the development of the nervous system. The mechanisms of MeHg-induced neurotoxicity are associated with the disruption of cellular homeostasis, and include oxidative stress, loss of calcium homeostasis, and impaired protein quality control. The stress inducible protein 1 (STI-1) is involved in the regulation of protein quality control by acting as a protein cochaperone to maintain optimal protein unfolding and refolding. Here, we utilized the Caenorhabditis elegans (C. elegans) model of MeHg toxicity to characterize the role of the sti-1 gene in MeHg-induced toxicity. We showed that lifespan and developmental milestone timings were significantly altered in sti-1 knockout (KO) animals with MeHg exposure. However, knocking down sti-1 by RNAi did not result in an analogous effect for lifespan, but did still sensitize to delays in developmental milestone progression by acute MeHg, suggesting that insufficiency of sti-1 does not recapitulate all phenotypes of the null mutation. Furthermore, inhibition of ATP levels by MeHg exposure was modulated by sti-1. Considering that the skn-1/gst-4 pathway is highly involved in metal's toxicity, such pathway was also explored in our model. We showed that sti-1 mutant worms exhibited impaired capacity to upregulate the antioxidant genes skn-1/gst-4, highlighting a central role of sti-1 in modulating antioxidant response. Lastly, we showed that loss-of-function mutation in the rrf-3 gene, which encodes a putative RNA-directed RNA polymerase, has significant effect in altering MeHg-induced toxicity by potentiating the animal's detoxification system. Altogether, our novel data show an indispensable role of protein quality control in the defense against MeHg toxicity.

Phytomelatonin as a signaling molecule for protein quality control via chaperone, autophagy, and ubiquitin–proteasome systems in plants

Physiological effects mediated by melatonin are attributable to its potent antioxidant activity as well as its role as a signaling molecule in inducing a vast array of melatonin-mediated genes. Here, we propose melatonin as a signaling molecule essential for protein quality control (PQC) in plants. PQC occurs by the coordinated activities of three systems: the chaperone network, autophagy, and the ubiquitin-proteasome system. With regard to the melatonin-mediated chaperone pathway, melatonin increases thermotolerance by induction of heat shock proteins and confers endoplasmic reticulum stress tolerance by increasing endoplasmic reticulum chaperone proteins. In chloroplasts, melatonin-induced chaperones, including Clps and CpHSP70s, play key roles in the PQC of chloroplast-localized proteins, such as Lhcb1, Lhcb4, and RBCL, during growth. Melatonin regulates PQC by autophagy processes, in which melatonin induces many autophagy (ATG) genes and autophagosome formation under stress conditions. Finally, melatonin-mediated plant stress tolerance is associated with up-regulation of stress-induced transcription factors, which are regulated by the ubiquitin-proteasome system. In this review, we propose that melatonin plays a pivotal role in PQC and consequently functions as a pleiotropic molecule under non-stress and adverse conditions in plants.

Tributyltin inhibits autophagy by decreasing lysosomal acidity in SH-SY5Y cells

Tributyltin (TBT) is an environmental pollutant that remains in marine sediments and is toxic to mammals. For example, TBT elicits neurotoxic and immunosuppressive effects on rats. However, it is not entirely understood how TBT causes toxicity. Autophagy plays a pivotal role in protein quality control and eliminates aggregated proteins and damaged organelles. We previously reported that TBT dephosphorylates mammalian target of rapamycin (mTOR), which may be involved in enhancement of autophagosome synthesis, in primary cultures of cortical neurons. Autophagosomes can accumulate due to enhancement of autophagosome synthesis or inhibition of autophagic degradation, and we did not clarify whether TBT alters autophagic flux. Here, we investigated the mechanism by which TBT causes accumulation of autophagosomes in SH-SY5Y cells. TBT inhibited autophagy without affecting autophagosome–lysosome fusion before it caused cell death. TBT dramatically decreased the acidity of lysosomes without affecting lysosomal membrane integrity. TBT decreased the mature protein level of cathepsin B, and this may be related to the decrease in lysosomal acidity. These results suggest that TBT inhibits autophagic degradation by decreasing lysosomal acidity. Autophagy impairment may be involved in the mechanism underlying neuronal death and/or T-cell-dependent thymus atrophy induced by TBT.

Perspective: Modulating the integrated stress response to slow aging and ameliorate age-related pathology.

Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. The Integrated Stress Response (ISR) is activated by diverse stimuli, leading to phosphorylation of the eukaryotic translation initiation factor elF2 in its α-subunit. Under replete conditions, elF2 orchestrates 5' cap-dependent mRNA translation and is thus responsible for general protein synthesis. elF2α phosphorylation, the key event of the ISR, reduces global mRNA translation while enhancing the expression of a signature set of stress response genes. Despite the critical role of protein quality control in healthy aging and in numerous longevity pathways, the role of the ISR in longevity remains largely unexplored. ISR activity increases with age, suggesting a potential link with the aging process. Although decreased protein biosynthesis, which occurs during ISR activation, have been linked to lifespan extension, recent data show that lifespan is limited by the ISR as its inhibition extends survival in nematodes and enhances cognitive function in aged mice. Here we survey how aging affects the ISR, the role of the ISR in modulating aging, and pharmacological interventions to tune the ISR. Finally, we will explore the ISR as a plausible target for clinical interventions in aging and age-related disease.

Conserved and Unique Roles of Chaperone-Dependent E3 Ubiquitin Ligase CHIP in Plants.

Protein quality control (PQC) is essential for maintaining cellular homeostasis by reducing protein misfolding and aggregation. Major PQC mechanisms include protein refolding assisted by molecular chaperones and the degradation of misfolded and aggregated proteins using the proteasome and autophagy. A C-terminus of heat shock protein (Hsp) 70-interacting protein [carboxy-terminal Hsp70-interacting protein (CHIP)] is a chaperone-dependent and U-box-containing E3 ligase. CHIP is a key molecule in PQC by recognizing misfolded proteins through its interacting chaperones and targeting their degradation. CHIP also ubiquitinates native proteins and plays a regulatory role in other cellular processes, including signaling, development, DNA repair, immunity, and aging in metazoans. As a highly conserved ubiquitin ligase, plant CHIP plays an important role in response to a broad spectrum of biotic and abiotic stresses. CHIP protects chloroplasts by coordinating chloroplast PQC both outside and inside the important photosynthetic organelle of plant cells. CHIP also modulates the activity of protein phosphatase 2A (PP2A), a crucial component in a network of plant signaling, including abscisic acid (ABA) signaling. In this review, we discuss the structure, cofactors, activities, and biological function of CHIP with an emphasis on both its conserved and unique roles in PQC, stress responses, and signaling in plants.

Methionine Redox Homeostasis in Protein Quality Control.

Bacteria live in different environments and are subject to a wide variety of fluctuating conditions. During evolution, they acquired sophisticated systems dedicated to maintaining protein structure and function, especially during oxidative stress. Under such conditions, methionine residues are converted into methionine sulfoxide (Met-O) which can alter protein function. In this review, we focus on the role in protein quality control of methionine sulfoxide reductases (Msr) which repair oxidatively protein-bound Met-O. We discuss our current understanding of the importance of Msr systems in rescuing protein function under oxidative stress and their ability to work in coordination with chaperone networks. Moreover, we highlight that bacterial chaperones, like GroEL or SurA, are also targeted by oxidative stress and under the surveillance of Msr. Therefore, integration of methionine redox homeostasis in protein quality control during oxidative stress gives a complete picture of this bacterial adaptive mechanism.

Protein quality control and regulated proteolysis in the genome-reduced organism Mycoplasma pneumoniae.

Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.

Aggresomal sequestration and STUB1-mediated ubiquitylation during mammalian proteaphagy of inhibited proteasomes

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.

Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis-associated protein variant superoxide dismutase 1 (SOD1)-A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.

Structural and functional properties of proteins interacting with small heat shock proteins.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones found in all domains of life, possessing significant roles in protein quality control in cells and assisting the refolding of non-native proteins. They are efficient chaperones against many in vitro protein substrates. Nevertheless, the in vivo native substrates of sHsps are not known. To better understand the functions of sHsps and the mechanisms by which they enhance heat resistance, sHsp-interacting proteins were identified using affinity purification under heat shock conditions. This paper aims at providing some insights into the characteristics of natural substrate proteins of sHsps. It seems that sHsps of prokaryotes, as well as sHsps of some eukaryotes, can bind to a wide range of substrate proteins with a preference for certain functional classes of proteins. Using Drosophila melanogaster mitochondrial Hsp22 as a model system, we observed that this sHsp interacted with the members of ATP synthase machinery. Mechanistically, Hsp22 interacts with the multi-type substrate proteins under heat shock conditions as well as non-heat shock conditions.

Оценка эффективности превентивной терапии с индуктором шаперонов U133 в модели доклинической стадии болезни Паркинсона у пожилых крыс

Parkinson's disease (PD) is a chronic steadily progressing neurodegenerative disease with a predominant lesion of nigrostriatal dopamine (DA-)ergic brain neurons. PD is an incurable disorder, which mostly affects the elderly. Misfolding and hyperphosphorylation of the α-synuclein protein, as well as the development of chronic neuroinflammation, are the main links in the pathogenesis of neurodegeneration in PD. Pharmacotherapy aimed at mobilizing the stress-inducible chaperone Hsp70, which plays a key role in protein quality control and possess immunomodulating activity, seems to be promising for the development of preventive therapy for PD. In the present study, we used a model of the preclinical stage of PD in elderly rats created by intranasal administration of a proteasome inhibitor lactacystin. A therapy based on the systemic administration of the chaperone inducer Hsp70, a low molecular weight quinoid compound U133 (acetylated 2,3,7-tris-O-glucoside echinochrome), was applied in the model of PD. We showed that the compound U133 causes a delayed increase in the Hsp70 content in the substantia nigra pars compacta (SNpc) in elderly animals. Preventive Hsp70-inducing therapy with U133 in the model of preclinical stage of PD in elderly rats weakened the process of neurodegeneration in SNpc and counteracted the development of neuroinflammation. At the same time, there was a decrease in the amount of aggregated α-synuclein and regression of post-translationally modified α-synuclein, phosphorylated at Ser129. The data obtained indicate that the prodrug, the small molecule, derivative of echinochrome U133 has significant therapeutic potential in the Parkinson-like pathology in elderly age. The results of the study are of scientific and practical importance for the development of innovative technology for preventive PD pharmacotherapy based on the domestic drug U133.

Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions.

DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and β-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric β-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure β-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded β-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and β-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric β-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Generation of MuRF-GFP transgenic zebrafish models for investigating murf gene expression and protein localization in Smyd1b and Hsp90α1 knockdown embryos.

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos.

Walk the Line: The Role of Ubiquitin in Regulating Transcription in Myocytes.

The ubiquitin-proteasome offers novel targets for potential therapies with their specific activities and tissue localization. Recently, the expansion of our understanding of how ubiquitin ligases (E3s) specifically regulate transcription has demonstrated their roles in skeletal muscle, complementing their roles in protein quality control and protein degradation. This review focuses on skeletal muscle E3s that regulate transcription factors critical to myogenesis and the maintenance of skeletal muscle wasting diseases.