Folding Chaperones Research Articles

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by β1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

A Lenalidomide-Inducible Suicide Switch for Gene- and Cell-Based Therapy

Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, have demonstrated promising efficacy against hematologic malignancies and liquid tumors but run the risk of severe life-threatening toxicities. Moreover, further modifications to enhance cellular immunotherapy potency are being tested to overcome treatment failures due to T cell dysfunction or suppression in the tumor microenvironment. Consequently, for novel and/or highly active cellular immunotherapies the inclusion of “suicide switches” may be prudent to suppress treatment-related adverse events by inducing specific depletion of the engineered cells. The clinical deployment of existing cell therapy suicide switches has been limited in part by imperfect performance characteristics, reliance on non-FDA-approved controller drugs, and the potential immunogenicity of non-human sequences. Herein, we report the engineering of a clinically suitable cell therapy suicide switch activated by targeted protein degradation, and the credentialing of this system to rapidly and irreversibly deplete CAR T cells in vitro and in vivo. We and others have previously demonstrated that lenalidomide-inducible degron tags can be linked to proteins of interest to allow for their selective degradation (Jan et al, Science Translational Medicine, 2021; Carbonneau et al, Cell Chemical Biology, 2021; Koduri et al, PNAS, 2019). Lenalidomide acts as a “molecular glue” that recruits neosubstrate proteins to the CRL4CRBN E3 ubiquitin ligase to be ubiquitinated and subsequently degraded by the proteasome. Caspase-activated DNase (CAD) and its inhibitor (ICAD) are broadly expressed pro- and anti-apoptotic proteins, respectively. ICAD serves as a chaperone for CAD folding and sequesters CAD activity by forming an inactive heterodimer with CAD. Upon apoptosis signaling, activated Caspase 3 cleaves ICAD, liberating CAD to form an active homodimer that acts as a pair of “molecular scissors” to create double strand breaks in the genome. We hypothesized that the overexpression of CAD and an ICAD-degron fusion protein at a stoichiometric 1:1 ratio could be well-tolerated, and furthermore, that lenalidomide treatment would deplete the ICAD-degron protein, thereby freeing CAD to cause cell death. After iterative optimization of promoter strength, transgene order, and degron placement, we evaluated an optimized ICAD-degron-CAD lenalidomide suicide switch versus inducible Caspase 9 in primary human T cells. The lenalidomide suicide switch induced more complete cell depletion versus iCasp9, and maximal cell depletion was seen with subtherapeutic nanomolar concentrations of lenalidomide. In multi-day co-culture live cell imaging assays with co-transduced and sorted CAR suicide switch T cells, tumor cell cytolysis and CAR T cell proliferation were comparable with or without expression of the suicide switch. Lenalidomide addition rapidly inhibited tumor cell cytolysis and depleted the suicide switch CAR T cells without subsequent re-expansion. In an in vivo NSG murine xenograft model with JeKo-1 tumor cell engraftment, suicide switch and control CAR T cells demonstrated comparable anti-tumor activity. For the suicide switch CAR T cells, tumor expansion accelerated after pomalidomide treatment, consistent with engineered cell suppression after suicide switch induction. We have also co-delivered the CAR and 2.2 kilobase suicide switch as a single multi-cistronic lentivector. In summary, a lenalidomide-inducible suicide switch composed of the stoichiometric pair of CAD and ICAD-degron proteins enabled rapid, near-complete engineered cell depletion without interfering with CAR T cell growth or effector functions in the models tested. Composed of all-human sequences and a controller available as a generic drug, the lenalidomide-inducible suicide switch is a clinically suitable system that may have broad applications to safeguard the development of highly potent investigational cellular immunotherapies.

Stem cells from human exfoliated deciduous teeth rejuvenate the liver in naturally aged mice by improving ribosomal and mitochondrial proteins

Background aimsAging is accompanied by a decline in cellular proteome homeostasis, mitochondrial, and metabolic function. Mesenchymal stromal cell (MSC) therapies have been reported to extend lifespan and delay some age-related pathologies, yet the anti-aging rate and mechanisms remain unclear. Here, we investigated the effects and mechanism by transplantation of stem cells from human exfoliated deciduous teeth (SHED) into the naturally aged mice model. MethodsSHED were cultured in vitro and injected into mice by caudal vein. The in vivo imaging uncovered that SHED labeled by DiR dye mainly migrated to the liver, spleen, and lung organs of wild-type mice. As the main metabolic organ and SHED homing place, the liver was selected for proteomics and aging clock algorithm (LiverClock) analysis, which was constructed to estimate the proteomic pattern related to liver age state. ResultsAfter 6 months of continuous SHED injections, the liver proteomic pattern was reversed from senescent (∼30 months) to a youthful state (∼3 months), accompanied with upregulation of hepatocytes marker genes, anti-aging protein Klotho, a global improvement of liver functional pathways proteins, and a dramatic regulation of ribosomal and mitochondrial proteins, including upregulation of translation elongation and ribosome-sparing proteins Rpsa and Rplp0; elongation factors Eif4a1, Eef1b2, Eif5a; protein-folding chaperones Hsp90aa and Hspe1; ATP synthesis proteins Atp5b, Atp5o, Atp5j; and downregulation of most ribosomal proteins, suggesting that the proteome homeostasis destruction and mitochondria dysfunction in the aged mice liver might be relieved after SHED treatment. ConclusionsSHED treatment could dramatically relieve the senescent state of the aged liver, affect ribosome component proteins and upregulate the ribosomal biogenesis proteins in the aged mice liver. These results may help understand the improvements and mechanisms of SHED treatment in anti-aging.

Detection and Characterisation of Endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Elaeidobius kamerunicus (Coleoptera: Curculionoidea), Pollinating Agent of Oil Palm, and Its Relationships between Populations.

Elaeidobius kamerunicus is the most efficient pollinator of oil palm. Wolbachia is an endosymbiotic bacteria associated with E. kamerunicus that has a potential to affect the fecundity and fitness of the E. kamerunicus. Despite their importance, no studies have been conducted to investigate its prevalence in E. kamerunicus. The objectives of this study were to detect and characterise Wolbachia in E. kamerunicus and determine the phylogenetic relationship of Wolbachia strains that infect E. kamerunicus by using three genetic markers namely Filamenting temperature-sensitive mutant Z (ftsZ), Chaperonin folding protein (groEL), and Citrate Synthase Coding Gene (gltA). DNA was extracted from 210 individuals of E. kamerunicus and the Wolbachia infections were detected using the wsp marker. The infected samples (n = 25, 11.9%) were then sequenced using ftsZ, gltA and groEL markers for strain characterization. In this study, a combination of four markers was used to construct the phylogeny of Wolbachia. Similar topologies were shown in all trees; Neighbour-Joining (NJ), Maximum Parsimony (MP), and Bayesian Inference (BI), which showed the mixing of individuals that harbor Wolbachia between populations. Interestingly, Wolbachia on E. kamerunicus was claded together with the species Drosophila simulans under supergroup B. This is the first report of Wolbachia infecting E. kamerunicus which is very valuable and significant as one of the parameters to evaluate the quality of the E. kamerunicus population for sustaining its function as a great pollinator for oil palm.

ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis.

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane-localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone-substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3-AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3-AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.

Abstract P2066: Forced Exercise Prevents Reductive Stress Induced Cardiomyocyte Remodeling And Cardiac Dysfunction

Background: Disruption of the endoplasmic reticulum (ER) function has been recognizedas a clinical signature of congestive heart failure, ischemia, and atherosclerosis. Whilepro-oxidative redox milieu is vital for the ER to regulate protein folding events. Recently,we reported that overabundant antioxidants in the heart cause reductive stress (RS) andimpair the protein folding mechanisms due to unresolved ER stress. Here, we testedwhether forced exercise prevents RS and improves ER function through augmenting ROSsignaling, thereby improving the PQC mechanisms in the heart. Methods: Cardiac-specific constitutively active Nrf2 (CaNrf2-TG) and non-transgenic (NTG) mice at 5-6weeks of age were subjected to forced treadmill exercise (FTE) for 20 weeks (15meters/minute with a 5° slope; for 45 minutes/day; for 6 days every week). Cardiacstructure and function were analyzed before and after the exercise. At 22 weeks, micewere sacrificed for analysis to assess the benefits of FTE. Results: RNA sequencingrevealed significant downregulation of key protein folding genes and chaperones in TGmice in association with the pathological remodeling at 6 months. Particularly, Pdi, Atf6, Calr, and Ero1α were downregulated (>2 fold) at 24 weeks in TG mice, indicating impairedER function in the remodeling hearts. These changes were coupled with increasedcardiomyocyte size and hypertrophy. Upon FTE, we observed significant improvementsin the structure and function of the cardiomyocytes. WGA staining showed the cross-section area of the cardiomyocytes was significantly reduced in the exercise groupcompared to the sedentary-TG mice experiencing cellular hypertrophy (115.7μm 2 NTGvs 428.8μm 2 TG vs 202.5 μm 2 TG-FTE). Echocardiography analysis demonstratedpreservation of cardiac structure and function (EF, FS, IVD, IVS, EDV, and ESV).Specifically, compensatory improvement in diastolic function (MVE/A ratio) was observedin TG-FTE exe group. Additionally, we observed that the ubiquitination of proteins wassignificantly reduced in the exercise group indicating deaccumulation of the misfoldedproteins in RS hearts. Conclusion: Together these results demonstrate that chronicexercise could ameliorate RS-induced ER stress and cardiac dysfunction.

The Role of Molecular Chaperones in Polypeptide Folding

Purpose: The aim of the study is to examine the role of molecular chaperones in polypeptide.
 Methodology: This study adopted a desktop methodology. This study used secondary data from which include review of existing literature from already published studies and reports that was easily accessed through online journals and libraries.
 Findings: The study revealed that molecular chaperones assist in the folding of polypeptides by stabilizing intermediate folding states and preventing the aggregation of unfolded or misfolded proteins. Molecular chaperones are crucial in preventing protein misfolding and aggregation-associated diseases, particularly in the context of neurodegenerative disorders
 Unique Contribution to Theory, Practice and Policy: The study was anchored on The Kinetic Partitioning Model was Proposed by Susan L. Lindquist and Levinthal's Paradox and the Hierarchical Folding Model and was proposed by Cyrus Levinthal. The study recommended support funding initiatives and research grants that focus on investigating the role of molecular chaperones in protein folding and their implications in various fields, including biotechnology, medicine, and drug discovery. Promote interdisciplinary research efforts to address the challenges and opportunities associated with chaperone-mediated folding.

Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA

Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 β subunit (eIF2β). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.

HtpG Is a Metal-Dependent Chaperone Which Assists the DnaK/DnaJ/GrpE Chaperone System of Mycobacterium tuberculosis via Direct Association with DnaJ2.

Protein folding is a crucial process in maintaining protein homeostasis, also known as proteostasis, in the cell. The requirement for the assistance of molecular chaperones in the appropriate folding of several proteins has already called into question the previously held view of spontaneous protein folding. These chaperones are highly ubiquitous cellular proteins, which not only help in mediating the proper folding of other nascent polypeptides but are also involved in refolding of the misfolded or the aggregated proteins. Hsp90 family proteins such as high-temperature protein G (HtpG) are abundant and ubiquitously expressed in both eukaryotic and prokaryotic cells. Although HtpG is known as an ATP-dependent chaperone protein in most organisms, function of this protein remains obscured in mycobacterial pathogens. Here, we aim to investigate significance of HtpG as a chaperone in the physiology of Mycobacterium tuberculosis. We report that M. tuberculosis HtpG (mHtpG) is a metal-dependent ATPase which exhibits chaperonin activity towards denatured proteins in coordination with the DnaK/DnaJ/GrpE chaperone system via direct association with DnaJ2. Increased expression of DnaJ1, DnaJ2, ClpX, and ClpC1 in a ΔhtpG mutant strain further suggests cooperativity of mHtpG with various chaperones and proteostasis machinery in M. tuberculosis. IMPORTANCE M. tuberculosis is exposed to variety of extracellular stressful conditions and has evolved mechanisms to endure and adapt to the adverse conditions for survival. mHtpG, despite being dispensable for M. tuberculosis growth under in vitro conditions, exhibits a strong and direct association with DnaJ2 cochaperone and assists the mycobacterial DnaK/DnaJ/GrpE (KJE) chaperone system. These findings suggest the potential role of mHtpG in stress management of the pathogen. Mycobacterial chaperones are responsible for folding of nascent protein as well as reactivation of protein aggregates. M. tuberculosis shows differential adaptive response subject to the availability of mHtpG. While its presence facilitates improved protein refolding via stimulation of the KJE chaperone activity, in the absence of mHtpG, M. tuberculosis enhances expression of DnaJ1/J2 cochaperones as well as Clp protease machinery for maintenance of proteostasis. Overall, this study provides a framework for future investigation to better decipher the mycobacterial proteostasis network in the light of stress adaptability and/or survival.

HSP90.2 modulates 2Q2-mediated wheat resistance against powdery mildew.

Wheat (Triticum aestivum L.) is a critical food crop feeding the world, but pathogens threaten its production. Wheat Heat Shock Protein 90.2 (HSP90.2) is a pathogen-inducible molecular chaperone folding nascent preproteins. Here, we used wheat HSP90.2 to isolate clients regulated at the posttranslational level. Tetraploid wheat hsp90.2 knockout mutant was susceptible to powdery mildew, while the HSP90.2 overexpression line was resistant, suggesting that HSP90.2 was essential for wheat resistance against powdery mildew. We next isolated 1500 clients of HSP90.2, which contained a wide variety of clients with different biological classifications. We utilized 2Q2, a nucleotide-binding leucine repeat-rich protein, as a model to investigate the potential of HSP90.2 interactome in fungal resistance. The transgenic line co-suppressing 2Q2 was more susceptible to powdery mildew, suggesting 2Q2 as a novel Pm-resistant gene. The 2Q2 protein resided in chloroplasts, and HSP90.2 played a critical role in the accumulation of 2Q2 in thylakoids. Our data provided over 1500 HSP90.2 clients with a potential regulation at the protein folding process and contributed a nontypical approach to isolate pathogenesis-related proteins.

HSP90.2 promotes CO2 assimilation rate, grain weight and yield in wheat.

Wheat fixes CO2 by photosynthesis into kernels to nourish humankind. Improving the photosynthesis rate is a major driving force in assimilating atmospheric CO2 and guaranteeing food supply for human beings. Strategies for achieving the above goal need to be improved. Here, we report the cloning and mechanism of CO2 ASSIMILATION RATE AND KERNEL-ENHANCED 1 (CAKE1) from durum wheat (Triticum turgidum L. var. durum). The cake1 mutant displayed a lower photosynthesis rate with smaller grains. Genetic studies identified CAKE1 as HSP90.2-B, encoding cytosolic molecular chaperone folding nascent preproteins. The disturbance of HSP90.2 decreased leaf photosynthesis rate, kernel weight (KW) and yield. Nevertheless, HSP90.2 over-expression increased KW. HSP90.2 recruited and was essential for the chloroplast localization of nuclear-encoded photosynthesis units, for example PsbO. Actin microfilaments docked on the chloroplast surface interacted with HSP90.2 as a subcellular track towards chloroplasts. A natural variation in the hexaploid wheat HSP90.2-B promoter increased its transcription activity, enhanced photosynthesis rate and improved KW and yield. Our study illustrated an HSP90.2-Actin complex sorting client preproteins towards chloroplasts to promote CO2 assimilation and crop production. The beneficial haplotype of Hsp90.2 is rare in modern varieties and could be an excellent molecular switch promoting photosynthesis rate to increase yield in future elite wheat varieties.

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris.

Fumonisins (FBs) are mycotoxins that threaten public health and food safety worldwide. Enzymatic degradation of Fumonisin B1 (FB1) through decarboxylation has attracted much attention, whereas application of FB1 carboxylesterase in detoxification requires more effective expression of the recombinant carboxylesterase. In this study, the carboxylesterase FumDM from Sphingopyxis sp. ASAG22 was codon-optimized and co-expressed with five different molecular chaperones (PDI, CPR5, ERO1, HAC1, and Bip) in order to improve the expression level of FumDM in Pichia pastoris (also known as Komagataella phaffii) GS115. The co-expression of different chaperones caused varying degrees of improvement in FumDM activity for FB1. The enzyme activities of recombinant strains over-expressing PDI and CPR5 reached the highest levels of 259.47 U/mL and 161.34 U/mL, 635% and 357% higher than the original enzyme activity, respectively. Transcriptomic analysis of the two recombinant strains in comparison with the control strain showed that the correct folding of proteins assisted by molecular chaperones played a key role in the improvement of FumDM expression and its enzyme activity. This study demonstrated that co-expression of carboxylesterase FumDM and folding chaperones was an efficient strategy and therefore might inspire new perspectives on the improvement of carboxylesterase for detoxification of FB1.

The bacterial secretosome for bacterial envelope biogenesis.

The Gram-negative bacterial envelope is a robust line of defence against physical and chemical environmental stress, including antibiotics. Therefore, its biogenesis is of considerable interest, as well as a target to address the growing problem of antimicrobial resistance. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machineries. The Sec translocon, a ubiquitous integral inner-membrane complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed into the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm, or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core hetero-trimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein transport and quality control. My talk will overview recent work on the association of the translocons of the inner- (Sec) and outer-membrane (BAM), along with periplasmic chaperones for the formation of the bacterial secretosome. I will describe our findings on the structure, dynamics and orchestration of this inter-membrane assembly for efficient passage of proteins into the inner-membrane, periplasm and outer-membrane - critical for efficient envelope biogenesis.

Reductionist insights into how chaperone charge influences RNA folding.

Non-coding RNAs are composed of modular secondary and tertiary structural units that themselves must form complex 3D structures for the entire RNA to fold and perform its biological function. However, the high density of negative charges associated with the phosphate backbone gives rise to substantial amounts of electrostatic repulsion, contributing to the large energetic barrier between unfolded and folded states. RNA folding chaperones (RFCs) are proteins that help lower this energy barrier by transiently interacting with RNA. Recent work has shown that some RFCs can function as macromolecular counterions for their RNA clients, thereby screening backbone electrostatic repulsion. However, the fundamental principles associated with how RFCs assist folding is largely unexplored. Here, we reduce RFCs to their smallest cationic constituents and then systematically “re-build” RFCs from these components (e.g., NH4+→ Lys→ polyK10→ RFC). We use single-molecule FRET to determine how these counterions influence both the relative population abundance and mean FRET efficiency of the folded and unfolded states of small model RNAs. From these studies, we have begun to determine aspects of RFCs that contribute to chaperoning RNA folding, such as net cationic charge and charge distribution. Here, we show that cationic amino acids (e.g., Lys and Arg) bind to RNA with apparent Kd values (appKd) on the order of 100 mM, which are comparable to those associated with their characteristic functional groups (e.g., ammonium and guanidinium). Further, increasing the net cationic charge of a counterion systematically decreases the appKd over several orders of magnitude. We also show a correlation between the decrease in appKd and a decrease in the folding free energy barrier. We then compare our observations of these polypeptide chaperones to the behavior of known RFCs (e.g., protamine, H1, and HCV nucleocapsid protein) with the small model RNAs.

Zinc-finger protein Zpr1 is a bespoke chaperone essential for eEF1A biogenesis

The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

Nucleotide Exchange Factors for Hsp70 Molecular Chaperones: GrpE, Hsp110/Grp170, HspBP1/Sil1, and BAG Domain Proteins.

Molecular chaperones of the Hsp70 family are key components of the cellular protein-folding machinery. Substrate folding is accomplished by iterative cycles of ATP binding, hydrolysis, and release. The ATPase activity of Hsp70 is regulated by two main classes of cochaperones: J-domain proteins stimulate ATPase hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) facilitate the conversion from the ADP-bound to the ATP-bound state, thus closing the chaperone folding cycle. NEF function can additionally be antagonized by ADP dissociation inhibitors. Beginning with the discovery of the prototypical bacterial NEF, GrpE, a large diversity of nucleotide exchange factors for Hsp70 have been identified, connecting it to a multitude of cellular processes in the eukaryotic cell. Here we review recent advances toward structure and function of nucleotide exchange factors from the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families and discuss how these cochaperones connect protein folding with cellular quality control and degradation pathways.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

A bacterial secretosome for regulated envelope biogenesis and quality control?

The Gram-negative bacterial envelope is the first line of defence against environmental stress and antibiotics. Therefore, its biogenesis is of considerable fundamental interest, as well as a challenge to address the growing problem of antimicrobial resistance. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed into the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm, or delivered to the β-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core hetero-trimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this 'secretosome' -a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these, and other factors, act to ensure efficient envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. We believe this organisation is critical for cell wall biogenesis and remodelling and thus its perturbation could be a means for the development of anti-microbials.

Pathway engineering facilitates efficient protein expression in Pichia pastoris.

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. KEY POINTS: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated.