Role In Folding Research Articles

Cracking the chaperone code through the computational microscope.

The heat shock protein 90 kDa (Hsp90) chaperone machinery plays a crucial role in maintaining cellular homeostasis. Beyond its traditional role in protein folding, Hsp90 is integral to key pathways influencing cellular function in health and disease. Hsp90 operates through the modular assembly of large multiprotein complexes, with their composition, stability, and localization adapting to the cell's needs. Its functional dynamics are finely tuned by ligand binding and post-translational modifications (PTMs). Here, we discuss how to disentangle the intricacies of the complex code that governs the crosstalk between dynamics, binding, PTMs, and the functions of the Hsp90 machinery using computer-based approaches. Specifically, we outline the contributions of computational and theoretical methods to the understanding of Hsp90 functions, ranging from providing atomic-level insights into its dynamics to clarifying the mechanisms of interactions with protein clients, cochaperones, and ligands. The knowledge generated in this framework can be actionable for the design and development of chemical tools and drugs targeting Hsp90 in specific disease-associated cellular contexts. Finally, we provide our perspective on how computation can be integrated into the study of the fine-tuning of functions in the highly complex Hsp90 landscape, complementing experimental methods for a comprehensive understanding of this important chaperone system.

The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.

Protein disulfide isomerase (PDI) is a member of the mercaptan isomerase family, primarily located in the endoplasmic reticulum (ER). At least 21 PDI family members have been identified. PDI plays a key role in protein folding, correcting misfolded proteins, and catalyzing disulfide bond formation, rearrangement, and breaking. It also acts as a molecular chaperone. Dysregulation of PDI activity is thus linked to diseases such as cancer, infections, immune disorders, thrombosis, neurodegenerative diseases, and metabolic disorders. In particular, elevated intracellular PDI levels can enhance cancer cell proliferation, metastasis, and invasion, making it a potential cancer marker. Cancer cells require extensive protein synthesis, with disulfide bond formation by PDI being a critical producer. Thus, cancer cells have higher PDI levels than normal cells. Targeting PDI can induce ER stress and activate the Unfolded Protein Response (UPR) pathway, leading to cancer cell apoptosis. This review discusses the structure and function of PDI, PDI inhibitors in cancer therapy, and the limitations of current inhibitors, proposing especially future directions for developing new PDI inhibitors.

ThermoLink: Bridging disulfide bonds and enzyme thermostability through database construction and machine learning prediction.

Disulfide bonds, covalently formed by sulfur atoms in cysteine residues, play a crucial role in protein folding and structure stability. Considering their significance, artificial disulfide bonds are often introduced to enhance protein thermostability. Although an increasing number of tools can assist with this task, significant amounts of time and resources are often wasted owing to inadequate consideration. To enhance the accuracy and efficiency of designing disulfide bonds for protein thermostability improvement, we initially collected disulfide bond and protein thermostability data from extensive literature sources. Thereafter, we extracted various sequence- and structure-based features and constructed machine-learning models to predict whether disulfide bonds can improve protein thermostability. Among all models, the neighborhood context model based on the Adaboost-DT algorithm performed the best, yielding "area under the receiver operating characteristic curve" and accuracy scores of 0.773 and 0.714, respectively. Furthermore, we also found AlphaFold2 to exhibit high superiority in predicting disulfide bonds, and to some extent, the coevolutionary relationship between residue pairs potentially guided artificial disulfide bond design. Moreover, several mutants of imine reductase 89 (IR89) with artificially designed thermostable disulfide bonds were experimentally proven to be considerably efficient for substrate catalysis. The SS-bond data have been integrated into an online server, namely, ThermoLink, available at guolab.mpu.edu.mo/thermoLink.

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Quantification of CH and NH/π-Stacking Interactions in Cells Using Nuclear Magnetic Resonance Spectroscopy.

π-Stacking, a type of noncovalent interactions involving aromatic residues, plays an important role in protein folding and function. In this work, an attempt has been made to measure CH/π and NH/π stacking interactions in a protein in Escherichia coli cells using a combined double-mutant cycle and nuclear magnetic resonance spectroscopy method. The results show that the CH/π and NH/π stacking interactions are generally weaker in cells than those in the buffer. The transient intermolecular noncovalent interactions between the protein and the complex cellular environment may compete with and thus weaken the stacking interactions in the protein. The weakening of stacking interactions can enhance the local conformational opening of proteins in E. coli cells. This is evident from the faster rates of amide hydrogen/deuterium exchange observed in cells than in the buffer, for residues that undergo local conformational opening. This study highlights the influence of the cellular environment on π-stacking and the conformational dynamics of proteins.

Alzheimer's disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity.

CD2-Associated protein (CD2AP) is a candidate susceptibility gene for Alzheimer's disease, but its role in the mammalian central nervous system remains largely unknown. We show that CD2AP protein is broadly expressed in the adult mouse brain, including within cortical and hippocampal neurons, where it is detected at pre-synaptic terminals. Deletion of Cd2ap altered dendritic branching and spine density, and impaired ubiquitin-proteasome system activity. Moreover, in mice harboring either one or two copies of a germline Cd2ap null allele, we noted increased paired-pulse facilitation at hippocampal Schaffer-collateral synapses, consistent with a haploinsufficient requirement for pre-synaptic release. Whereas conditional Cd2ap knockout in the brain revealed no gross behavioral deficits in either 3.5- or 12-month-old mice, Cd2ap heterozygous mice demonstrated subtle impairments in discrimination learning using a touchscreen task. Based on unbiased proteomics, partial or complete loss of Cd2ap triggered perturbation of proteins with roles in protein folding, lipid metabolism, proteostasis, and synaptic function. Overall, our results reveal conserved, dose-sensitive requirements for CD2AP in the maintenance of neuronal structure and function, including synaptic homeostasis and plasticity, and inform our understanding of possible cell-type specific mechanisms in Alzheimer's Disease.

Cyclophilin inhibition as a strategy for the treatment of human disease.

Cyclophilins (Cyps), characterized as peptidyl-prolyl cis-trans isomerases (PPIases), are highly conserved and ubiquitous, playing a crucial role in protein folding and cellular signaling. This review summarizes the biochemical pathways mediated by Cyps, including their involvement in pathological states such as viral replication, inflammation, and cancer progression, to underscore the therapeutic potential of Cyp inhibition. The exploration of Cyp inhibitors (CypI) in this review, particularly non-immunosuppressive cyclosporine A (CsA) derivatives, highlights their significance as therapeutic agents. The structural and functional nuances of CsA derivatives are examined, including their efficacy, mechanism of action, and the balance between therapeutic benefits and off-target effects. The landscape of CypI is evaluated to emphasize the clinical need for targeted approaches to exploit the complex biology of Cyps and to propose future directions for research that may enhance the utility of non-immunosuppressive CsA derivatives in treating diseases where Cyps play a key pathological role.

Epsilon subunit of T-complex protein-1 from Leishmania donovani: A tetrameric chaperonin

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite.

Computational analysis of propeptide-containing proteins and prediction of their post-cleavage conformation changes.

A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and activation and are present in about 2.3% of reviewed proteins in the UniProt database. They are often found in secreted or membrane-bound proteins including proteolytic enzymes, hormones, and toxins. We identified a variety of globular and nonglobular Pfam domains in protein sequences designated as propeptides, some of which form intramolecular interactions with other domains in the mature proteins. Propeptide-containing enzymes mostly function as proteases, as they are depleted in other enzyme classes such as hydrolases acting on DNA and RNA, isomerases, and lyases. We applied AlphaFold to generate structural models for over 7000 proteins with propeptides having no less than 20 residues. Analysis of residue contacts in these models revealed conformational changes for over 300 proteins before and after the cleavage of the propeptide. Examples of conformation change occur in several classes of proteolytic enzymes in the families of subtilisins, trypsins, aspartyl proteases, and thermolysin-like metalloproteases. In most of the observed cases, cleavage of the propeptide releases the constraints imposed by the covalent bond between the propeptide and the mature protein, and cleavage enables stronger interactions between the propeptide and the mature protein. These findings suggest that post-cleavage propeptides could play critical roles in regulating the activity of mature proteins.

Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E.coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E.coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Chaperone regulation of biomolecular condensates

Biomolecular condensation allows for the dynamic organization of molecules in time and space. Condensate formation is regulated through many mechanisms including the action of molecular chaperones. While molecular chaperones have long been viewed through the lens of their roles in protein folding, misfolding, and quality control, their ability to manipulate protein-protein interactions is increasingly recognized to play a major role in the precise control of condensate biology. In this review we highlight recent studies investigating the roles of canonical and non-canonical chaperones in regulating condensate formation, material state, and dispersal. We discuss the broadening of longstanding conceptions of chaperone functions to include condensate regulation, and the discovery of previously unappreciated chaperone activities in well-known proteins. We close by considering the biological activities being uncovered during the ongoing upheaval at the boundary between chaperone biology and biomolecular condensation.

Unraveling the Connection: Pain and Endoplasmic Reticulum Stress.

Pain is a complex and multifaceted experience. Recent research has increasingly focused on the role of endoplasmic reticulum (ER) stress in the induction and modulation of pain. The ER is an essential organelle for cells and plays a key role in protein folding and calcium dynamics. Various pathological conditions, such as ischemia, hypoxia, toxic substances, and increased protein production, may disturb protein folding, causing an increase in misfolding proteins in the ER. Such an overload of the folding process leads to ER stress and causes the unfolded protein response (UPR), which increases folding capacity in the ER. Uncompensated ER stress impairs intracellular signaling and cell function, resulting in various diseases, such as diabetes and degenerative neurological diseases. ER stress may be a critical universal mechanism underlying human diseases. Pain sensations involve the central as well as peripheral nervous systems. Several preclinical studies indicate that ER stress in the nervous system is enhanced in various painful states, especially in neuropathic pain conditions. The purpose of this narrative review is to uncover the intricate relationship between ER stress and pain, exploring molecular pathways, implications for various pain conditions, and potential therapeutic strategies.

Impact of Hydrodynamic Interactions on the Kinetic Pathway of Protein Folding.

Protein folding is a fundamental process critical to cellular function and human health, but it remains a grand challenge in biophysics. Hydrodynamic interaction (HI) plays a vital role in the self-organization of soft and biological materials, yet its role in protein folding is not fully understood despite folding occurring in a fluid environment. Here, we use the fluid particle dynamics method to investigate many-body hydrodynamic couplings between amino acid residues and fluid motion in the folding kinetics of a coarse-grained four-α-helices bundle protein. Our results reveal that HI helps select fast folding pathways to the native state without being kinetically trapped, significantly speeding up the folding kinetics compared to its absence. First, the directional flow along the protein backbone expedites protein collapse. Then, the incompressibility-induced squeezing flow effects retard the accumulation of non-native hydrophobic contacts, thus preventing the protein from being trapped in local energy minima during the conformational search of the native structure. We also find that the significance of HI in folding kinetics depends on temperature, with a pronounced effect under biologically relevant conditions. Our findings suggest that HI, particularly the short-range squeezing effect, may be crucial in avoiding protein misfolding.

Heat shock protein paradigms in cancer progression: future therapeutic perspectives.

Heat-shock proteins (HSPs), also known as stress proteins, are ubiquitously present in all forms of life. They play pivotal roles in protein folding and unfolding, the formation of multiprotein complexes, the transportation and sorting of proteins into their designated subcellular compartments, the regulation of the cell cycle, and signalling processes. These HSPs encompass HSP27, HSP40, HSP70, HSP60, and HSP90, each contributing to various cellular functions. In the context of cancer, HSPs exert influence by either inhibiting or activating diverse signalling pathways, thereby impacting growth, differentiation, and cell division. This article offers an extensive exploration of the functions of HSPs within the realms of pharmacology and cancer biology. HSPs are believed to play substantial roles in the mechanisms underlying the initiation and progression of cancer. They hold promise as valuable clinical markers for cancer diagnosis, potential targets for therapeutic interventions, and indicators of disease progression. In times of cellular stress, HSPs function as molecular chaperones, safeguarding the structural and functional integrity of proteins and aiding in their proper folding. Moreover, HSPs play a crucial role in cancer growth, by regulating processes such as angiogenesis, cell proliferation, migration, invasion, and metastasis.

Evolutionary trade-off between heat shock resistance, growth at high temperature, and virulence expression in Salmonella Typhimurium.

Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.

Molecular evolution of the heat shock protein family and the role of HSP30 in immune response and wound healing in lampreys (Lethenteron reissneri)

Heat shock proteins (HSPs) are molecular chaperones that ubiquitously exist in various organisms and play essential roles in protein folding, transport, and expression. While most HSPs are highly conserved across species, a few HSPs are evolutionarily distinct in some species and may have unique functions. To explore the evolutionary history of the vertebrate HSP family, we identify members of the HSP family at the genome-wide level in lampreys (Lethenteron reissneri), a living representative of jawless vertebrates diverged from jawed vertebrates over 500 million years ago. The phylogenetic analysis reveals that the lamprey HSP family contains HSP90a1, HSP90a2, HSC70, HSP60, HSP30, HSP27, HSP17, and HSP10, which have a primitive status in the molecular evolution of vertebrate HSPs. Transcriptome analysis reveals the expression distribution of members of the HSP family in various tissues of lampreys. It is shown that HSP30, normally found in birds, amphibians, and fish, is also present in lampreys, with remarkable expansion of HSP30 gene copies in the lamprey genome. The transcription of HSP30 is significantly induced in leukocytes and heart of lampreys during various pathogens or poly(I:C) stimulation, indicating that HSP30 may be involved in the immune defense of lampreys in response to bacterial or viral infection. Immunohistochemistry demonstrates significantly increased HSP30 expression in subcutaneous muscle tissue after skin injury in lamprey models of wound repair. Furthermore, transcriptome analysis shows that ectopic expression of HSP30 in 3T3-L1 fibroblasts affect the expression of genes related to the PI3K-AKT signaling pathway, suggesting that HSP30 could serves as a negative regulator of fibrosis. These results indicate that HSP30 may play a critical role in facilitating the process of lamprey skin repair following injury. This study provides new insights into the origin and evolution of the HSP gene family in vertebrates and offers valuable clues to reveal the important role of HSP30 in immune defense and wound healing of lampreys.

The glycosylation landscape of prostate cancer tissues and biofluids.

An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.

Analysis of Protein Glycosylation in the ER.

Protein N-glycosylation is an essential posttranslational modification which is initiated in the endoplasmic reticulum (ER). In plants, the N-glycans play a pivotal role in protein folding and quality control. Through the interaction of glycan processing and binding reactions mediated by ER-resident glycosidases and specific carbohydrate-binding proteins, the N-glycans contribute to the adoption of a native protein conformation. Properly folded glycoproteins are released from these processes and allowed to continue their transit to the Golgi where further processing and maturation of N-glycans leads to the formation of more complex structures with different functions. Incompletely folded glycoproteins are removed from the ER by a highly conserved degradation process to prevent the accumulation or secretion of misfolded proteins and maintain ER homeostasis. Here, we describe methods to analyze the N-glycosylation status and the glycan-dependent ER-associated degradation process in plants.

The role of protein folding in prenucleation clusters on the activity of enzyme@metal–organic frameworks

Two steps must be satisfied to achieve high activity enzyme@MOFs: proper enzyme folding and low MOF crystallinity.

Glycosort: A Computational Solution to Post-process Quantitative Large-Scale Intact Glycopeptide Analyses.

Protein glycosylation is a post-translational modification involving the addition of carbohydrates to proteins and plays a crucial role in protein folding and various biological processes such as cell recognition, differentiation, and immune response. The vast array of natural sugars available allows the generation of plenty of unique glycan structures in proteins, adding complexity to the regulation and biological functions of glycans. The diversity is further increased by enzymatic site preferences and stereochemical conjugation, leading to an immense amount of different glycan structures. Understanding glycosylation heterogeneity is vital for unraveling the impact of glycans on different biological functions. Evaluating site occupancies and structural heterogeneity aids in comprehending glycan-related alterations in biological processes. Several software tools are available for large-scale glycoproteomics studies; however, integrating identification and quantitative data to assess heterogeneity complexity often requires extensive manual data processing. To address this challenge, we present a python script that automates the integration of Byonic and MaxQuant outputs for glycoproteomic data analysis. The script enables the calculation of site occupancy percentages by glycans and facilitates the comparison of glycan structures and site occupancies between two groups. This automated tool offers researchers a means to organize and interpret their high-throughput quantitative glycoproteomic data effectively.