Protein Quality Control Machinery Research Articles

CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Remodeling of the ribosomal quality control and integrated stress response by viral ubiquitin deconjugases

The strategies adopted by viruses to reprogram the translation and protein quality control machinery and promote infection are poorly understood. Here, we report that the viral ubiquitin deconjugase (vDUB)—encoded in the large tegument protein of Epstein-Barr virus (EBV BPLF1)—regulates the ribosomal quality control (RQC) and integrated stress responses (ISR). The vDUB participates in protein complexes that include the RQC ubiquitin ligases ZNF598 and LTN1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of the 40 S particle and inhibits the degradation of translation-stalled polypeptides by the proteasome. Impairment of the RQC correlates with the readthrough of stall-inducing mRNAs and with activation of a GCN2-dependent ISR that redirects translation towards upstream open reading frames (uORFs)- and internal ribosome entry sites (IRES)-containing transcripts. Physiological levels of active BPLF1 promote the translation of the EBV Nuclear Antigen (EBNA)1 mRNA in productively infected cells and enhance the release of progeny virus, pointing to a pivotal role of the vDUB in the translation reprogramming that enables efficient virus production.

Transcriptional Regulators of Plant Adaptation to Heat Stress.

Heat stress (HS) is becoming an increasingly large problem for food security as global warming progresses. As sessile species, plants have evolved different mechanisms to cope with the disruption of cellular homeostasis, which can impede plant growth and development. Here, we summarize the mechanisms underlying transcriptional regulation mediated by transcription factors, epigenetic regulators, and regulatory RNAs in response to HS. Additionally, cellular activities for adaptation to HS are discussed, including maintenance of protein homeostasis through protein quality control machinery, and autophagy, as well as the regulation of ROS homeostasis via a ROS-scavenging system. Plant cells harmoniously regulate their activities to adapt to unfavorable environments. Lastly, we will discuss perspectives on future studies for improving urban agriculture by increasing crop resilience to HS.

Reduction of Nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration.

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding TDP-43, account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS (fALS)-causing mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer's disease, and Parkinson's disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a novel function of Nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and lifespan deficits in two distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared along the autophagy-lysosome axis, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

Protein quality control machinery supports primary ciliogenesis by eliminating GDP-bound Rab8-family GTPases

The small GTPase Rab8 plays a vital role in the vesicular trafficking of cargo proteins from the trans-Golgi network to target membranes. Upon reaching its target destination, Rab8 is released from the vesicular membrane into the cytoplasm via guanosine triphosphate (GTP) hydrolysis. The fate of GDP-bound Rab8 released from the destination membranes, however, has not been investigated adequately. In this study, we found that GDP-bound Rab8 subfamily proteins are targeted for immediate degradation, and the pre-emptive quality control machinery is responsible for eliminating these proteins in a nucleotide-specific manner. We provide evidence that components of this quality control machinery have a critical role in vesicular trafficking events, including the formation of primary cilia, a process regulated by the Rab8 subfamily. These results suggest that the protein degradation machinery plays a critical role in the integrity of membrane trafficking by limiting the excessive accumulation of GDP-bound Rab8 subfamily proteins.

Functional, pathogenic, and pharmacological roles of protein folding intermediates.

Protein expression and function in eukaryotic cells are tightly harmonized processes modulated by the combination of different layers of regulation, including transcription, processing, stability, and translation of messenger RNA, as well as assembly, maturation, sorting, recycling, and degradation of polypeptides. Integrating all these pathways and the protein quality control machinery, deputed to avoid the production and accumulation of aberrantly folded proteins, determines protein homeostasis. Over the last decade, the combined development of accurate time-resolved experimental techniques and efficient computer simulations has opened the possibility of investigating biological mechanisms at atomic resolution with physics-based models. A meaningful example is the reconstruction of protein folding pathways at atomic resolution, which has enabled the characterization of the folding kinetics of biologically relevant globular proteins consisting of a few hundred amino acids. Combining these innovative computational technologies with rigorous experimental approaches reveals the existence of non-native metastable states transiently appearing along the folding process of such proteins. Here, we review the primary evidence indicating that these protein folding intermediates could play roles in disparate biological processes, from the posttranslational regulation of protein expression to disease-relevant protein misfolding mechanisms. Finally, we discuss how the information encoded into protein folding pathways could be exploited to design an entirely new generation of pharmacological agents capable of promoting the selective degradation of protein targets.

Identification of subfunctionalized aggregate-remodeling J-domain proteins in Arabidopsis thaliana.

J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.

Intersecting PTMS regulate clearance of pathogenic tau by the CHIP ubiquitin ligase

AbstractBackgroundBiochemical and cellular models of tau post‐translational modifications (PTMs) were generated to understand how pathogenic tau species evade the protein quality control machinery during the pathogenesis of neurodegeneration.MethodA pathogenic fragment of 0N4R tau representing the species derived from caspase‐cleavage at Asp421 (tauC3) bearing native PTMs was purified from Sf9 insect cells, and its binding to the ubiquitin ligase CHIP was analyzed by ELISA. Differential scanning fluorimetry and fluorescence polarization assays were used to identify relevant sites of modification that alter the affinity of CHIP for tauC3. X‐ray crystallography was employed to define the molecular details of the interaction of the CHIP TPR domain with tauC3.ResultPhosphorylation of tauC3 was shown to reduce the affinity of the CHIP‐tauC3 interaction and attenuate ubiquitination of tauC3 by CHIP. Phosphorylation of tauC3 at Ser416 was shown to be sufficient to significantly reduce the affinity of CHIP‐tauC3 interaction and promote tauC3 accumulation in cells. Introducing specific mutations into CHIP rescued binding to phosphorylated tauC3 and improved ubiquitination and clearance of tauC3.ConclusionC‐terminal phosphorylation at Ser416 of caspase‐cleaved tau inhibits tau clearance by the CHIP ubiquitin ligase and represents a targetable modality to promote clearance of pathogenic tau species during the pathogenesis of neurodegenerative diseases.

Dynamic quality control machinery that operates across compartmental borders mediates the degradation of mammalian nuclear membrane proteins.

Many human diseases are caused by mutations in nuclear envelope (NE) proteins. How protein homeostasis and disease etiology are interconnected at the NE is poorly understood. Specifically, the identity of local ubiquitin ligases that facilitate ubiquitin-proteasome-dependent NE protein turnover is presently unknown. Here, we employ a short-lived, Lamin B receptor disease variant as a model substrate in a genetic screen to uncover key elements of NE protein turnover. We identify the ubiquitin-conjugating enzymes (E2s) Ube2G2 and Ube2D3, the membrane-resident ubiquitin ligases (E3s) RNF5 and HRD1, and the poorly understood protein TMEM33. RNF5, but not HRD1, requires TMEM33 both for efficient biosynthesis and function. Once synthesized, RNF5 responds dynamically to increased substrate levels at the NE by departing from the endoplasmic reticulum, where HRD1 remains confined. Thus, mammalian protein quality control machinery partitions between distinct cellular compartments to address locally changing substrate loads, establishing a robust cellular quality control system.

245-LB: An Ankyrin-B–Mediated Protein Chaperone Mechanism Modulates Pancreatic ß-Cell Susceptibility to Proteotoxicity-Induced ER Stress and Apoptosis

Impaired insulin secretion contributes to the development and progression of diabetes. β-cells initially cope with the genetic, environmental, and diabetogenic stresses that diminish insulin secretion or the peripheral response to it by increasing insulin production. However, sustained increases in the insulin precursor proinsulin (PI) place additional demand on the secretory and protein quality control (PQC) machineries of β-cells, triggering endoplasmic reticulum (ER) and oxidative stress, mitochondrial dysfunction, and β-cell death. Thus, understanding the mechanisms that regulate β-cell responses to increased insulin secretory requirements is critical to prevent β-cell failure. We reported that the cytoskeletal scaffolding protein ankyrin-B (AnkB) regulates insulin secretion by maintaining normal levels of the inositol-1,4,5-triphosphate receptor (IP3R) at the ER and intracellular calcium in β-cells. IP3R levels and insulin secretion were impaired in knock-in mice carrying the p.R1788W AnkB (AnkB-RW) variant associated with type 2 diabetes (T2D) in Caucasians and Hispanics. Here, we show that selective loss of AnkB or expression of the T2D AnkB-RW variant lead to reduced insulin secretion and accumulation of PI and islet specific amyloid polypeptide in β-cells. These changes are accompanied by enhanced induction of the maladaptive unfolded protein response and apoptosis. Interestingly, the T2D AnkB-RW variant lies within the binding site for the protein chaperone Dnajb1 (Hsp40) . Our results indicate that the Dnajb1-Hspa1a complex, which recognizes and delivers protein clients to Hsp70 chaperones to promote proper folding, refolding, or degradation, is downregulated in β-cells from β-cell-specific AnkB knockout or AnkB-RW mice. Increased proteotoxicity and reduced insulin secretion were also observed upon knockdown of Dnajb1 in rat INS1 832/13 cells. Together, our findings suggest that AnkB and its integration into the Dnajb1-Hspa1a pathway modulates PQC and promotes β-cell survival under diabetogenic conditions. Disclosure J. Tzeng: None. H. Hohmeier: None. D. Lorenzo: None.

Folding state as the Decision Maker in Detecting Misfolded Proteins by Hsp70

Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C‐terminus of Hsc70‐interacting protein (CHIP). Hsp70 itself helps misfolded “client” proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP‐mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above‐mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate‐binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide‐locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.

Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell’s protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.

A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress.

Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.

Biosensors for Studying Neuronal Proteostasis.

Cellular health depends on the integrity and functionality of the proteome. Each cell is equipped with a protein quality control machinery that maintains protein homeostasis (proteostasis) by helping proteins adopt and keep their native structure, and ensuring the degradation of damaged proteins. Postmitotic cells such as neurons are especially vulnerable to disturbances of proteostasis. Defects of protein quality control occur in aging and have been linked to several disorders, including neurodegenerative diseases. However, the exact nature and time course of such disturbances in the context of brain diseases remain poorly understood. Sensors that allow visualization and quantitative analysis of proteostasis capacity in neurons are essential for gaining a better understanding of disease mechanisms and for testing potential therapies. Here, I provide an overview of available biosensors for assessing the functionality of the neuronal proteostasis network, point out the advantages and limitations of different sensors, and outline their potential for biological discoveries and translational applications.

Protein chain collapse modulation and folding stimulation by GroEL-ES.

The collapse of polypeptides is thought important to protein folding, aggregation, intrinsic disorder, and phase separation. However, whether polypeptide collapse is modulated in cells to control protein states is unclear. Here, using integrated protein manipulation and imaging, we show that the chaperonin GroEL-ES can accelerate the folding of proteins by strengthening their collapse. GroEL induces contractile forces in substrate chains, which draws them into the cavity and triggers a general compaction and discrete folding transitions, even for slow-folding proteins. This collapse enhancement is strongest in the nucleotide-bound states of GroEL and is aided by GroES binding to the cavity rim and by the amphiphilic C-terminal tails at the cavity bottom. Collapse modulation is distinct from other proposed GroEL-ES folding acceleration mechanisms, including steric confinement and misfold unfolding. Given the prevalence of collapse throughout the proteome, we conjecture that collapse modulation is more generally relevant within the protein quality control machinery.

Differential Interactions of Molecular Chaperones and Yeast Prions.

Baker’s yeast Saccharomyces cerevisiae is an important model organism that is applied to study various aspects of eukaryotic cell biology. Prions in yeast are self-perpetuating heritable protein aggregates that can be leveraged to study the interaction between the protein quality control (PQC) machinery and misfolded proteins. More than ten prions have been identified in yeast, of which the most studied ones include [PSI+], [URE3], and [PIN+]. While all of the major molecular chaperones have been implicated in propagation of yeast prions, many of these chaperones differentially impact propagation of different prions and/or prion variants. In this review, we summarize the current understanding of the life cycle of yeast prions and systematically review the effects of different chaperone proteins on their propagation. Our analysis clearly shows that Hsp40 proteins play a central role in prion propagation by determining the fate of prion seeds and other amyloids. Moreover, direct prion-chaperone interaction seems to be critically important for proper recruitment of all PQC components to the aggregate. Recent results also suggest that the cell asymmetry apparatus, cytoskeleton, and cell signaling all contribute to the complex network of prion interaction with the yeast cell.

Heat Shock Factors in Protein Quality Control and Spermatogenesis.

Proper regulation of cellular protein quality control is crucial for cellular health. It appears that the protein quality control machinery is subjected to distinct regulation in different cellular contexts such as in somatic cells and in germ cells. Heat shock factors (HSFs) play critical role in the control of quality of cellular proteins through controlling expression of many genes encoding different proteins including those for inducible protein chaperones. Mammalian cells exert distinct mechanism of cellular functions through maintenance of tissue-specific HSFs. Here, we have discussed different HSFs and their functions including those during spermatogenesis. We have also discussed the different heat shock proteins induced by the HSFs and their activities in those contexts. We have also identified several small molecule activators and inhibitors of HSFs from different sources reported so far.

Structural basis of DegP protease temperature-dependent activation.

Protein quality control is an essential cellular function mainly executed by a vast array of different proteases and molecular chaperones. One of the bacterial high temperature requirement A (HtrA) protein family members, the homo-oligomeric DegP protease, plays a crucial role in the Escherichia coli protein quality control machinery by removing unfolded proteins or preventing their aggregation and chaperoning them to their final folded state within the periplasm. DegP contains two regulatory PDZ domains, which play key roles in substrate recognition and in the transformation of DegP between inactive hexameric and proteolytic active cage-like structures. Here, we analyze the interaction and dynamics of the DegP PDZ domains underlying this transformation by high-resolution NMR spectroscopy complemented with biochemical cleavage assays. We identify an interdomain molecular lock, which controls the interactions between the two PDZ domains, regulated by fine-tuned temperature-dependent protein dynamics, and which is potentially conserved in proteins harboring tandem PDZ domains.