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Cold Spring Harbor Perspectives in... Jan 2020The proteasome, the most complex protease known, degrades proteins that have been conjugated to ubiquitin. It faces the unique challenge of acting enzymatically on... (Review)
Review
The proteasome, the most complex protease known, degrades proteins that have been conjugated to ubiquitin. It faces the unique challenge of acting enzymatically on hundreds and perhaps thousands of structurally diverse substrates, mechanically unfolding them from their native state and translocating them vectorially from one specialized compartment of the enzyme to another. Moreover, substrates are modified by ubiquitin in myriad configurations of chains. The many unusual design features of the proteasome may have evolved in part to endow this enzyme with a robust ability to process substrates regardless of their identity. The proteasome plays a major role in preserving protein homeostasis in the cell, which requires adaptation to a wide variety of stress conditions. Modulation of proteasome function is achieved through a large network of proteins that interact with it dynamically, modify it enzymatically, or fine-tune its levels. The resulting adaptability of the proteasome, which is unique among proteases, enables cells to control the output of the ubiquitin-proteasome pathway on a global scale.
Topics: Adenosine Triphosphate; Animals; Caenorhabditis elegans; Cryoelectron Microscopy; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation; Homeostasis; Humans; Models, Molecular; Molecular Conformation; Nuclear Respiratory Factor 1; Proteasome Endopeptidase Complex; Protein Denaturation; Protein Engineering; Protein Folding; Protein Processing, Post-Translational; Protein Transport; Saccharomyces cerevisiae Proteins; Transcription Factors; Ubiquitin; Ubiquitin Thiolesterase
PubMed: 30833452
DOI: 10.1101/cshperspect.a033985 -
Frontiers in Molecular Biosciences 2023The AAA+ ATPase p97 (also called VCP or Cdc48) is a major protein unfolding machine with hundreds of clients in diverse cellular pathways that are critical for cell... (Review)
Review
The AAA+ ATPase p97 (also called VCP or Cdc48) is a major protein unfolding machine with hundreds of clients in diverse cellular pathways that are critical for cell homeostasis, proliferation and signaling. In this review, we summarize recent advances in understanding how diverse client proteins are targeted to the p97 machine to facilitate client degradation or to strip clients from binding partners for regulation. We describe an elaborate system that is governed by at least two types of alternative adapters. The Ufd1-Npl4 adapter along with accessory adapters targets ubiquitylated clients in the majority of pathways and uses ubiquitin as a universal unfolding tag. In contrast, the family of SEP-domain adapters such as p37 can target clients directly to p97 in a ubiquitin-independent manner. Despite the different targeting strategies, both pathways converge by inserting the client into the p97 pore to initiate a peptide threading mechanism through the central channel of p97 that drives client protein unfolding, protein extraction from membranes and protein complex disassembly processes.
PubMed: 36825201
DOI: 10.3389/fmolb.2023.1142989 -
Analytical Chemistry May 2020Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass...
Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa /) and its performance capabilities and limits were explored for a range of protein and protein complexes. The results show new potential for this instrument platform for studies of large protein and protein complexes and provides a roadmap for extending the performance metrics for studies of even larger, more complex systems, namely, membrane protein complexes and their interactions with ligands.
Topics: Concanavalin A; Fructose-Bisphosphate Aldolase; Ion Mobility Spectrometry; Mass Spectrometry; Protein Conformation; Protein Unfolding; Streptavidin; Ubiquitin
PubMed: 32338885
DOI: 10.1021/acs.analchem.0c00772 -
Biophysical Journal Aug 2021The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger...
The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.
Topics: Entropy; Plant Proteins; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 34087209
DOI: 10.1016/j.bpj.2021.05.019 -
Redox Biology Feb 2020Methylglyoxal (MG) is a highly reactive aldehyde spontaneously formed in human cells mainly as a by-product of glycolysis. Such endogenous metabolite reacts with...
Methylglyoxal (MG) is a highly reactive aldehyde spontaneously formed in human cells mainly as a by-product of glycolysis. Such endogenous metabolite reacts with proteins, nucleotides and lipids forming advanced glycation end-products (AGEs). MG binds to arginine, lysine and cysteine residues of proteins causing the formation of stable adducts that can interfere with protein function. Among the proteins affected by glycation, MG has been found to react with superoxide dismutase 1 (SOD1), a fundamental anti-oxidant enzyme that is abundantly expressed in neurons. Considering the high neuronal susceptibility to MG-induced oxidative stress, we sought to investigate by mass spectrometry and NMR spectroscopy which are the structural modifications induced on SOD1 by the reaction with MG. We show that MG reacts preferentially with the disulfide-reduced, demetallated form of SOD1, gradually causing its unfolding, and to a lesser extent, with the intermediate state of maturation - the reduced, zinc-bound homodimer - causing its gradual monomerization. These results suggest that MG could impair the correct maturation of SOD1 in vivo, thus both increasing cellular oxidative stress and promoting the cytotoxic misfolding and aggregation process of SOD1.
Topics: Binding Sites; Glycolysis; Humans; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Oxidative Stress; Protein Binding; Protein Structure, Secondary; Protein Unfolding; Pyruvaldehyde; Superoxide Dismutase-1
PubMed: 31931282
DOI: 10.1016/j.redox.2019.101421 -
Proceedings of the National Academy of... Mar 2021Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical...
Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.
Topics: Humans; Kinetics; Protein Aggregates; Protein Unfolding; Structure-Activity Relationship; alpha-Synuclein
PubMed: 33649211
DOI: 10.1073/pnas.2012171118 -
Biophysical Journal Nov 2022Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed...
Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.
Topics: Tumor Suppressor Protein p53; Genes, p53; Kinetics; Mutation; Protein Unfolding; Protein Aggregates
PubMed: 36230002
DOI: 10.1016/j.bpj.2022.10.013 -
Proceedings of the National Academy of... Sep 2023The vanilloid receptor TRPV1 is an exquisite nociceptive sensor of noxious heat, but its temperature-sensing mechanism is yet to define. Thermodynamics dictate that this...
The vanilloid receptor TRPV1 is an exquisite nociceptive sensor of noxious heat, but its temperature-sensing mechanism is yet to define. Thermodynamics dictate that this channel must undergo an unusually energetic allosteric transition. Thus, it is of fundamental importance to measure directly the energetics of this transition in order to properly decipher its temperature-sensing mechanism. Previously, using submillisecond temperature jumps and patch-clamp recording, we estimated that the heat activation for TRPV1 opening incurs an enthalpy change on the order of 100 kcal/mol. Although this energy is on a scale unparalleled by other known biological receptors, the generally imperfect allosteric coupling in proteins implies that the actual amount of heat uptake driving the TRPV1 transition could be much larger. In this paper, we apply differential scanning calorimetry to directly monitor the heat flow in TRPV1 that accompanies its temperature-induced conformational transition. Our measurements show that heat invokes robust, complex thermal transitions in TRPV1 that include both channel opening and a partial protein unfolding transition and that these two processes are inherently coupled. Our findings support that irreversible protein unfolding, which is generally thought to be destructive to physiological function, is essential to TRPV1 thermal transduction and, possibly, to other strongly temperature-dependent processes in biology.
Topics: Biological Transport; Hot Temperature; Temperature; Thermodynamics; TRPV Cation Channels
PubMed: 37639609
DOI: 10.1073/pnas.2300305120 -
International Journal of Molecular... Dec 2020The effects of airway inflammation on airway smooth muscle (ASM) are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). In this review... (Review)
Review
The effects of airway inflammation on airway smooth muscle (ASM) are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). In this review article, we will provide a unifying hypothesis for a homeostatic response to airway inflammation that mitigates oxidative stress and thereby provides resilience to ASM. Previous studies have shown that acute exposure to TNFα increases ASM force generation in response to muscarinic stimulation (hyper-reactivity) resulting in increased ATP consumption and increased tension cost. To meet this increased energetic demand, mitochondrial O consumption and oxidative phosphorylation increases but at the cost of increased reactive oxygen species (ROS) production (oxidative stress). TNFα-induced oxidative stress results in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and mitochondria of ASM. In the ER, TNFα selectively phosphorylates inositol-requiring enzyme 1 alpha (pIRE1α) triggering downstream splicing of the transcription factor X-box binding protein 1 (XBP1s); thus, activating the pIRE1α/XBP1s ER stress pathway. Protein unfolding in mitochondria also triggers an unfolded protein response (UPR). In our conceptual framework, we hypothesize that activation of these pathways is homeostatically directed towards mitochondrial remodeling via an increase in peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression, which in turn triggers: (1) mitochondrial fragmentation (increased dynamin-related protein-1 (Drp1) and reduced mitofusin-2 (Mfn2) expression) and mitophagy (activation of the Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin mitophagy pathway) to improve mitochondrial quality; (2) reduced Mfn2 also results in a disruption of mitochondrial tethering to the ER and reduced mitochondrial Ca influx; and (3) mitochondrial biogenesis and increased mitochondrial volume density. The homeostatic remodeling of mitochondria results in more efficient O consumption and oxidative phosphorylation and reduced ROS formation by individual mitochondrion, while still meeting the increased ATP demand. Thus, the energetic load of hyper-reactivity is shared across the mitochondrial pool within ASM cells.
Topics: Animals; Homeostasis; Humans; Inflammation; Mitochondria; Muscle, Smooth; Organelle Biogenesis; Oxidative Stress; Oxygen Consumption; Protein Unfolding; Tumor Necrosis Factor-alpha; Unfolded Protein Response
PubMed: 33396378
DOI: 10.3390/ijms22010363 -
Communications Biology Apr 2020Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available,...
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.
Topics: Bacterial Toxins; Cell Membrane; Escherichia coli Proteins; Hemolysin Proteins; Kinetics; Membrane Potentials; Mutation; Protein Folding; Protein Transport; Protein Unfolding; Single Molecule Imaging; Structure-Activity Relationship; Thioredoxins
PubMed: 32246057
DOI: 10.1038/s42003-020-0841-4