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Biochemical Society Transactions Jun 2023Proteostasis and redox homeostasis are tightly interconnected and most protein quality control pathways are under direct redox regulation which allow cells to...
Proteostasis and redox homeostasis are tightly interconnected and most protein quality control pathways are under direct redox regulation which allow cells to immediately respond to oxidative stress conditions. The activation of ATP-independent chaperones serves as a first line of defense to counteract oxidative unfolding and aggregation of proteins. Conserved cysteine residues evolved as redox-sensitive switches which upon reversible oxidation induce substantial conformational rearrangements and the formation of chaperone-active complexes. In addition to harnessing unfolding proteins, these chaperone holdases interact with ATP-dependent chaperone systems to facilitate client refolding and restoring proteostasis during stress recovery. This minireview gives an insight into highly orchestrated mechanisms regulating the stress-specific activation and inactivation of redox-regulated chaperones and their role in cell stress responses.
Topics: Humans; Molecular Chaperones; Oxidation-Reduction; Cytoplasm; Proteostasis; Adenosine Triphosphate; Oxidative Stress
PubMed: 37140269
DOI: 10.1042/BST20221304 -
International Journal of Molecular... Feb 2021Aggrephagy is defined as the selective degradation of aggregated proteins by autophagosomes. Protein aggregation in organs and cells has been highlighted as a cause of... (Review)
Review
Aggrephagy is defined as the selective degradation of aggregated proteins by autophagosomes. Protein aggregation in organs and cells has been highlighted as a cause of multiple diseases, including neurodegenerative diseases, cardiac failure, and renal failure. Aggregates could pose a hazard for cell survival. Cells exhibit three main mechanisms against the accumulation of aggregates: protein refolding by upregulation of chaperones, reduction of protein overload by translational inhibition, and protein degradation by the ubiquitin-proteasome and autophagy-lysosome systems. Deletion of autophagy-related genes reportedly contributes to intracellular protein aggregation in vivo. Some proteins recognized in aggregates in preeclamptic placentas include those involved in neurodegenerative diseases. As aggregates are derived both intracellularly and extracellularly, special endocytosis for extracellular aggregates also employs the autophagy machinery. In this review, we discuss how the deficiency of aggrephagy and/or macroautophagy leads to poor placentation, resulting in preeclampsia or fetal growth restriction.
Topics: Animals; Female; Humans; Lysosomes; Macroautophagy; Placenta; Pre-Eclampsia; Pregnancy; Protein Aggregation, Pathological
PubMed: 33670947
DOI: 10.3390/ijms22052432 -
International Journal of Molecular... Jul 2020A delicate intracellular balance among protein synthesis, folding, and degradation is essential to maintaining protein homeostasis or proteostasis, and it is challenged... (Review)
Review
A delicate intracellular balance among protein synthesis, folding, and degradation is essential to maintaining protein homeostasis or proteostasis, and it is challenged by genetic and environmental factors. Molecular chaperones and the ubiquitin proteasome system (UPS) play a vital role in proteostasis for normal cellular function. As part of protein quality control, molecular chaperones recognize misfolded proteins and assist in their refolding. Proteins that are beyond repair or refolding undergo degradation, which is largely mediated by the UPS. The importance of protein quality control is becoming ever clearer, but it can also be a disease-causing mechanism. Diseases such as phenylketonuria (PKU) and hereditary tyrosinemia-I (HT1) are caused due to mutations in and gene, resulting in reduced protein stability, misfolding, accelerated degradation, and deficiency in functional proteins. Misfolded or partially unfolded proteins do not necessarily lose their functional activity completely. Thus, partially functional proteins can be rescued from degradation by molecular chaperones and deubiquitinating enzymes (DUBs). Deubiquitination is an important mechanism of the UPS that can reverse the degradation of a substrate protein by covalently removing its attached ubiquitin molecule. In this review, we discuss the importance of molecular chaperones and DUBs in reducing the severity of PKU and HT1 by stabilizing and rescuing mutant proteins.
Topics: Animals; Deubiquitinating Enzymes; Humans; Molecular Chaperones; Phenylketonurias; Proteasome Endopeptidase Complex; Protein Folding; Protein Stability; Proteolysis; Tyrosinemias; Ubiquitination
PubMed: 32679806
DOI: 10.3390/ijms21144996 -
Methods in Molecular Biology (Clifton,... 2023The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this...
The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatography etc).Recombinant proteins may be expressed in host cells as insoluble inclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins.In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein's propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures.
Topics: Cell Death; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Computational Biology
PubMed: 37647006
DOI: 10.1007/978-1-0716-3362-5_17 -
Chemical Science Nov 2019Interactions between proteins and surfactants are of relevance in many applications including food, washing powder formulations, and drug formulation. The anionic...
Interactions between proteins and surfactants are of relevance in many applications including food, washing powder formulations, and drug formulation. The anionic surfactant sodium dodecyl sulfate (SDS) is known to unfold globular proteins, while the non-ionic surfactant octaethyleneglycol monododecyl ether (CE) can be used to refold proteins from their SDS-denatured state. While unfolding have been studied in detail at the protein level, a complete picture of the interplay between protein and surfactant in these processes is lacking. This gap in our knowledge is addressed in the current work, using the β-sheet-rich globular protein β-lactoglobulin (bLG). We combined stopped-flow time-resolved SAXS, fluorescence, and circular dichroism, respectively, to provide an unprecedented in-depth picture of the different steps involved in both protein unfolding and refolding in the presence of SDS and CE. During unfolding, core-shell bLG-SDS complexes were formed within ∼10 ms. This involved an initial rapid process where protein and SDS formed aggregates, followed by two slower processes, where the complexes first disaggregated into single protein structures situated asymmetrically on the SDS micelles, followed by isotropic redistribution of the protein. Refolding kinetics (>100 s) were slower than unfolding (<30 s), and involved rearrangements within the mixing deadtime (∼5 ms) and transient accumulation of unfolded monomeric protein, differing in structure from the original bLG-SDS structure. Refolding of bLG involved two steps: extraction of most of the SDS from the complexes followed by protein refolding. These results reveal that surfactant-mediated unfolding and refolding of proteins are complex processes with rearrangements occurring on time scales from sub-milliseconds to minutes.
PubMed: 34123043
DOI: 10.1039/c9sc04831f -
The FEBS Journal Mar 2021Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss... (Review)
Review
Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss the inherent properties of multidomain proteins under a force vector from a structural and functional perspective. We then characterize three main systems where multidomain proteins could play major roles through mechanical unfolding: muscular contraction, cellular mechanotransduction, and bacterial adhesion. We analyze how key multidomain proteins for each system can produce a gain-of-function from the perspective of a fine-tuned quantized response, a molecular battery, delivery of mechanical work through refolding, elasticity tuning, protection and exposure of cryptic sites, and binding-induced mechanical changes. Understanding how mechanical unfolding and refolding affect function will have important implications in designing mechano-active drugs against conditions such as muscular dystrophy, cancer, or novel antibiotics.
Topics: Elasticity; Mechanotransduction, Cellular; Models, Molecular; Protein Binding; Protein Domains; Protein Folding; Protein Unfolding; Proteins; Stress, Mechanical; Thermodynamics
PubMed: 32761965
DOI: 10.1111/febs.15508 -
Frontiers in Molecular Biosciences 2023While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular... (Review)
Review
While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.
PubMed: 37021114
DOI: 10.3389/fmolb.2023.1155521 -
Biotechnology Letters Apr 2022To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its...
To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its derivative to act as a linear structure control (L-SRGFP). SRGFP and L-SRGFP showed similar fluorescence characteristics to the wild-type GFP, while compared with GFP and L-SRGFP, the thermal stability and denaturation resistance of SRGFP were improved. The refolding efficiencies of these three denatured proteins were investigated under different pH, temperature and initial protein concentration conditions, and it was found that SRGFP was superior to GFP and L-SRGFP in terms of refolding yield and refolding speed. In the pH range of 8.0-8.5, SRGFP could basically recover all fluorescence, while GFP and L-SRGFP recovered only about 87.52% and 88.58%. When refolded at a high temperature (37 °C), SRGFP still recovered 85.27% of the fluorescence, whereas GFP and L-SRGFP recovered only around 69.43% and 68.45%. At a high initial protein concentration (5 mg/mL), the refolding yield of SRGFP was about 15% higher than that of both GFP and L-SRGFP. These results suggest that the introduction of SpyRing structure (head-to-tail cyclization via SpyTag and SpyCatcher) improved the protein's stability and facilitated the refolding of denatured protein.
Topics: Cyclization; Green Fluorescent Proteins; Hot Temperature; Protein Denaturation; Temperature
PubMed: 35359178
DOI: 10.1007/s10529-022-03246-x -
Journal of Molecular Biology May 2020Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and...
Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics. We performed new targeted experiments and analyzed classical data, covering multiple types of non-ordered aggregates, chaperone combinations, and experimental conditions. We found that, irrespective of the behavior observed, the balance of surface disaggregation and refolding in solution universally determines the reactivation dynamics, which is broadly described by two characteristic times. This characterization makes it possible to use activity measurements to accurately infer the underlying loss of aggregated protein and to quantify, for the first time, the refolding rates of the soluble intermediates.
Topics: Benzothiazoles; Dynamic Light Scattering; Models, Molecular; Molecular Chaperones; Protein Aggregates; Protein Folding
PubMed: 32147456
DOI: 10.1016/j.jmb.2020.03.002 -
Trends in Biotechnology May 2020Recombinant proteins expressed as bacterial inclusion bodies (IBs) are now receiving tremendous attention for many diverse applications in the areas of industrial and... (Review)
Review
Recombinant proteins expressed as bacterial inclusion bodies (IBs) are now receiving tremendous attention for many diverse applications in the areas of industrial and medical biotechnology. Understanding the structure-function relationship of protein in IBs has recently created new possibilities in developing innovative isolation, solubilization, refolding, and purification processes for high-throughput recovery of bioactive protein from bacterial IBs. This opinion article describes the advantages, disadvantages, and major challenges presently associated with each of the processing steps. Finally, we conclude with the possible solutions for each operational step and the future direction of the basic and translational research to achieve maximum benefit from IB aggregates.
Topics: Biotechnology; Escherichia coli; Humans; Inclusion Bodies; Protein Folding; Recombinant Proteins; Structure-Activity Relationship
PubMed: 31954528
DOI: 10.1016/j.tibtech.2019.12.011