-
International Journal of Molecular... Jan 2022The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum... (Review)
Review
The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy . We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that Δ of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.
Topics: Algorithms; Kinetics; Models, Molecular; Models, Theoretical; Protein Conformation; Protein Folding; Protein Refolding; Protein Stability; Proteins; Proteome; Proteomics; Recombinant Proteins; Solubility; Species Specificity; Thermodynamics
PubMed: 35008947
DOI: 10.3390/ijms23010521 -
Frontiers in Aging 2022Cardiovascular disorder is the major health burden and cause of death among individuals worldwide. As the cardiomyocytes lack the ability for self-renewal, it is utmost... (Review)
Review
Cardiovascular disorder is the major health burden and cause of death among individuals worldwide. As the cardiomyocytes lack the ability for self-renewal, it is utmost necessary to surveil the protein quality in the cells. The Bcl-2 associated anthanogene protein (BAG) family and molecular chaperones (HSP70, HSP90) actively participate in maintaining cellular protein quality control (PQC) to limit cellular dysfunction in the cells. The BAG family contains a unique BAG domain which facilitates their interaction with the ATPase domain of the heat shock protein 70 (HSP70) to assist in protein folding. Among the BAG family members (BAG1-6), BAG5 protein is unique since it has five domains in tandem, and the binding of BD5 induces certain conformational changes in the nucleotide-binding domain (NBD) of HSP70 such that it loses its affinity for binding to ADP and results in enhanced protein refolding activity of HSP70. In this review, we shall describe the role of BAG5 in modulating mitophagy, endoplasmic stress, and cellular viability. Also, we have highlighted the interaction of BAG5 with other proteins, including PINK, DJ-1, CHIP, and their role in cellular PQC. Apart from this, we have described the role of BAG5 in cellular metabolism and aging.
PubMed: 35821856
DOI: 10.3389/fragi.2022.844168 -
International Journal of Molecular... Aug 2019The discovery of heat shock proteins shaped our view of protein folding in the cell. Since their initial discovery, chaperone proteins were identified in all domains of... (Review)
Review
The discovery of heat shock proteins shaped our view of protein folding in the cell. Since their initial discovery, chaperone proteins were identified in all domains of life, demonstrating their vital and conserved functional roles in protein homeostasis. Chaperone proteins maintain proper protein folding in the cell by utilizing a variety of distinct, characteristic mechanisms to prevent aberrant intermolecular interactions, prevent protein aggregation, and lower entropic costs to allow for protein refolding. Continued study has found that chaperones may exhibit alternative functions, including maintaining protein folding during endoplasmic reticulum (ER) import and chaperone-mediated degradation, among others. Alternative chaperone functions are frequently controlled by post-translational modification, in which a given chaperone can switch between functions through covalent modification. This review will focus on the Hsp70 class chaperones and their Hsp40 co-chaperones, specifically highlighting the importance of post-translational control of chaperones. These modifications may serve as a target for therapeutic intervention in the treatment of diseases of protein misfolding and aggregation.
Topics: Allosteric Regulation; Animals; HSP70 Heat-Shock Proteins; Humans; Protein Processing, Post-Translational
PubMed: 31466231
DOI: 10.3390/ijms20174207 -
Frontiers in Plant Science 2021Protein quality control (PQC) is essential for maintaining cellular homeostasis by reducing protein misfolding and aggregation. Major PQC mechanisms include protein... (Review)
Review
Protein quality control (PQC) is essential for maintaining cellular homeostasis by reducing protein misfolding and aggregation. Major PQC mechanisms include protein refolding assisted by molecular chaperones and the degradation of misfolded and aggregated proteins using the proteasome and autophagy. A C-terminus of heat shock protein (Hsp) 70-interacting protein [carboxy-terminal Hsp70-interacting protein (CHIP)] is a chaperone-dependent and U-box-containing E3 ligase. CHIP is a key molecule in PQC by recognizing misfolded proteins through its interacting chaperones and targeting their degradation. CHIP also ubiquitinates native proteins and plays a regulatory role in other cellular processes, including signaling, development, DNA repair, immunity, and aging in metazoans. As a highly conserved ubiquitin ligase, plant CHIP plays an important role in response to a broad spectrum of biotic and abiotic stresses. CHIP protects chloroplasts by coordinating chloroplast PQC both outside and inside the important photosynthetic organelle of plant cells. CHIP also modulates the activity of protein phosphatase 2A (PP2A), a crucial component in a network of plant signaling, including abscisic acid (ABA) signaling. In this review, we discuss the structure, cofactors, activities, and biological function of CHIP with an emphasis on both its conserved and unique roles in PQC, stress responses, and signaling in plants.
PubMed: 34305988
DOI: 10.3389/fpls.2021.699756 -
Nature Dec 2023Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory...
Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion. Spike opening exposes domain S1, allowing it to bind to proteinaceous receptors, and is also thought to enable protein refolding during membrane fusion. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1. This binding triggers the transition of S1 domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.
Topics: Humans; Allosteric Regulation; Betacoronavirus; Common Cold; Cryoelectron Microscopy; Molecular Dynamics Simulation; Polysaccharides; Protein Binding; Protein Conformation; Sialic Acids; Spike Glycoprotein, Coronavirus; Immune Evasion
PubMed: 37794193
DOI: 10.1038/s41586-023-06599-z -
Biomedicines Aug 2021Selective recognition and removal of faulty transcripts and misfolded polypeptides are crucial for cell viability. In eukaryotic cells, nonsense-mediated mRNA decay... (Review)
Review
Selective recognition and removal of faulty transcripts and misfolded polypeptides are crucial for cell viability. In eukaryotic cells, nonsense-mediated mRNA decay (NMD) constitutes an mRNA surveillance pathway for sensing and degrading aberrant transcripts harboring premature termination codons (PTCs). NMD functions also as a post-transcriptional gene regulatory mechanism by downregulating naturally occurring mRNAs. As NMD is activated only after a ribosome reaches a PTC, PTC-containing mRNAs inevitably produce truncated and potentially misfolded polypeptides as byproducts. To cope with the emergence of misfolded polypeptides, eukaryotic cells have evolved sophisticated mechanisms such as chaperone-mediated protein refolding, rapid degradation of misfolded polypeptides through the ubiquitin-proteasome system, and sequestration of misfolded polypeptides to the aggresome for autophagy-mediated degradation. In this review, we discuss how UPF1, a key NMD factor, contributes to the selective removal of faulty transcripts via NMD at the molecular level. We then highlight recent advances on UPF1-mediated communication between mRNA surveillance and protein quality control.
PubMed: 34440199
DOI: 10.3390/biomedicines9080995 -
Biomacromolecules Oct 2022Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely...
Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.
Topics: Alginates; Phosphoglycerate Kinase; Polymers; Protein Denaturation; Protein Folding; Protein Stability
PubMed: 36054903
DOI: 10.1021/acs.biomac.2c00297 -
International Journal of Biological... Apr 2023Protein folding is a biophysical process by which proteins reach a specific three-dimensional structure. The amino acid sequence of a polypeptide chain contains all the... (Review)
Review
Protein folding is a biophysical process by which proteins reach a specific three-dimensional structure. The amino acid sequence of a polypeptide chain contains all the information needed to determine the final three-dimensional structure of a protein. When producing a recombinant protein, several problems can occur, including proteolysis, incorrect folding, formation of inclusion bodies, or protein aggregation, whereby the protein loses its natural structure. To overcome such limitations, several strategies have been developed to address each specific issue. Identification of proper protein refolding conditions can be challenging, and to tackle this high throughput screening for different recombinant protein folding conditions can prove a sound solution. Different approaches have emerged to tackle refolding issues. One particular approach to address folding issues involves molecular chaperones, highly conserved proteins that contribute to proper folding by shielding folding proteins from other proteins that could hinder the process. Proper protein folding is one of the main prerequisites for post-translational modifications. Incorrect folding, if not dealt with, can lead to a buildup of protein misfoldings that damage cells and cause widespread abnormalities. Said post-translational modifications, widespread in eukaryotes, are critical for protein structure, function and biological activity. Incorrect post-translational protein modifications may lead to individual consequences or aggregation of therapeutic proteins. In this review article, we have tried to examine some key aspects of recombinant protein expression. Accordingly, the relevance of these proteins is highlighted, major problems related to the production of recombinant protein and to refolding issues are pinpointed and suggested solutions are presented. An overview of post-translational modification, their biological significance and methods of identification are also provided. Overall, the work is expected to illustrate challenges in recombinant protein expression.
Topics: Recombinant Proteins; Protein Folding; Molecular Chaperones; Protein Processing, Post-Translational; Proteolysis
PubMed: 36708896
DOI: 10.1016/j.ijbiomac.2023.123407 -
Journal of Molecular Biology Jan 2022The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and... (Review)
Review
The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.
Topics: Animals; Heat-Shock Proteins, Small; Humans; Molecular Chaperones; Multiprotein Complexes; Protein Aggregates; Protein Folding; Protein Multimerization; Proteostasis; Stress Granules
PubMed: 34271010
DOI: 10.1016/j.jmb.2021.167157 -
MBio Oct 2022Bacteria have evolved many different signal transduction systems to sense and respond to changing environmental conditions. Signal integration is mainly achieved by...
Bacteria have evolved many different signal transduction systems to sense and respond to changing environmental conditions. Signal integration is mainly achieved by signal recognition at extracytosolic ligand-binding domains (LBDs) of receptors. Hundreds of different LBDs have been reported, and our understanding of their sensing properties is growing. Receptors must function over a range of environmental pH values, but there is little information available on the robustness of sensing as a function of pH. Here, we have used isothermal titration calorimetry to determine the pH dependence of ligand recognition by nine LBDs that cover all major LBD superfamilies, of periplasmic solute-binding proteins, and cytosolic LBDs. We show that periplasmic LBDs recognize ligands over a very broad pH range, frequently stretching over eight pH units. This wide pH range contrasts with a much narrower pH response range of the cytosolic LBDs analyzed. Many LBDs must be dimeric to bind ligands, and analytical ultracentrifugation studies showed that the LBD of the Tar chemoreceptor forms dimers over the entire pH range tested. The pH dependences of Pseudomonas aeruginosa motility and chemotaxis were bell-shaped and centered at pH 7.0. Evidence for pH robustness of signaling was obtained by Förster Resonance Energy Transfer (FRET) measurements of the chemotaxis pathway responses in Escherichia coli. Bacteria have evolved several strategies to cope with extreme pH, such as periplasmic chaperones for protein refolding. The intrinsic pH resistance of periplasmic LBDs appears to be another strategy that permits bacteria to survive under adverse conditions. Demonstration of the pH robustness of extracytoplasmic sensing reveals a previously undescribed evolutionary mechanism that enables bacteria to monitor environmental changes under changing conditions. This mechanism includes the maintenance of the dimeric state of four-helixbundle ligand-binding domains (LBDs). The construction of biosensors is a rapidly growing field of research, and their use to monitor the progression of the COVID-19 pandemic has impressively demonstrated their usefulness. LBDs represent an enormous reservoir of binding modules that can be used to create novel biosensors. Among ligands recognized by LBDs are neurotransmitters, hormones, and quorum-sensing signals. The demonstration that extracytosolic LBDs bind their signals over a wide range of pH values will facilitate the design of biosensors that function under highly variable conditions of acidity and alkalinity.
Topics: Humans; Ligands; Bacterial Proteins; Protein Binding; Pandemics; COVID-19; Chemotaxis; Bacteria; Escherichia coli; Hormones; Hydrogen-Ion Concentration
PubMed: 36154178
DOI: 10.1128/mbio.01650-22