-
International Journal of Molecular... Jun 2024Heat stroke, a hazardous hyperthermia-related illness, is characterized by CNS injury, particularly long-lasting brain damage. A root cause for hyperthermic neurological...
Heat stroke, a hazardous hyperthermia-related illness, is characterized by CNS injury, particularly long-lasting brain damage. A root cause for hyperthermic neurological damage is heat-induced proteotoxic stress through protein aggregation, a known causative agent of neurological disorders. Stress magnitude and enduring persistence are highly correlated with hyperthermia-associated neurological damage. We used an untargeted proteomic approach using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and characterize time-series proteome-wide changes in dose-responsive proteotoxic stress models in medulloblastoma [Daoy], neuroblastoma [SH-SY5Y], and differentiated SH-SY5Y neuron-like cells [SH(D)]. An integrated analysis of condition-time datasets identified global proteome-wide differentially expressed proteins (DEPs) as part of the heat-induced proteotoxic stress response. The condition-specific analysis detected higher DEPs and upregulated proteins in extreme heat stress with a relatively conservative and tight regulation in differentiated SH-SY5Y neuron-like cells. Functional network analysis using ingenuity pathway analysis (IPA) identified common intercellular pathways associated with the biological processes of protein, RNA, and amino acid metabolism and cellular response to stress and membrane trafficking. The condition-wise temporal pathway analysis in the differentiated neuron-like cells detects a significant pathway, functional, and disease association of DEPs with processes like protein folding and protein synthesis, Nervous System Development and Function, and Neurological Disease. An elaborate dose-dependent stress-specific and neuroprotective cellular signaling cascade is also significantly activated. Thus, our study provides a comprehensive map of the heat-induced proteotoxic stress response associating proteome-wide changes with altered biological processes. This helps to expand our understanding of the molecular basis of the heat-induced proteotoxic stress response with potential translational connotations.
Topics: Humans; Neurons; Proteomics; Proteome; Cell Line, Tumor; Heat-Shock Response; Tandem Mass Spectrometry; Chromatography, Liquid; Cell Differentiation; Proteotoxic Stress
PubMed: 38928492
DOI: 10.3390/ijms25126787 -
International Journal of Molecular... Jun 2024The SH2 domains of SHP2 play a crucial role in determining the function of the SHP2 protein. While the folding and binding properties of the isolated NSH2 and CSH2...
The SH2 domains of SHP2 play a crucial role in determining the function of the SHP2 protein. While the folding and binding properties of the isolated NSH2 and CSH2 domains have been extensively studied, there is limited information about the tandem SH2 domains. This study aims to elucidate the folding and binding kinetics of the NSH2-CSH2 tandem domains of SHP2 through rapid kinetic experiments, complementing existing data on the isolated domains. The results indicate that while the domains generally fold and unfold independently, acidic pH conditions induce complex scenarios involving the formation of a misfolded intermediate. Furthermore, a comparison of the binding kinetics of isolated NSH2 and CSH2 domains with the NSH2-CSH2 tandem domains, using peptides that mimic specific portions of Gab2, suggests a dynamic interplay between NSH2 and CSH2 in binding Gab2 that modulate the microscopic association rate constant of the binding reaction. These findings, discussed in the context of previous research on the NSH2 and CSH2 domains, enhance our understanding of the function of the SH2 domain tandem of SHP2.
Topics: Protein Tyrosine Phosphatase, Non-Receptor Type 11; src Homology Domains; Protein Folding; Kinetics; Protein Binding; Humans; Hydrogen-Ion Concentration; Adaptor Proteins, Signal Transducing
PubMed: 38928272
DOI: 10.3390/ijms25126566 -
International Journal of Molecular... Jun 2024This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of... (Review)
Review
This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.
Topics: Water; Proteins; Protein Folding; Humans; Protein Binding; Protein Conformation; Ligands; Recombinant Proteins
PubMed: 38928046
DOI: 10.3390/ijms25126339 -
MSphere Jun 2024The genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly...
The genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops , we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of and commensal species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for infection and immune avoidance.
PubMed: 38926904
DOI: 10.1128/msphere.00450-24 -
Nature Communications Jun 2024As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is...
As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short is introduced. Analyzing a set of ene reductases, is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.
Topics: Solvents; Enzyme Stability; Protein Unfolding; Temperature; Transition Temperature; Oxidoreductases
PubMed: 38926341
DOI: 10.1038/s41467-024-49774-0 -
Journal of Chemical Theory and... Jun 2024Protein folding is a critical process that determines the functional state of proteins. Proper folding is essential for proteins to acquire their functional...
Protein folding is a critical process that determines the functional state of proteins. Proper folding is essential for proteins to acquire their functional three-dimensional structures and execute their biological role, whereas misfolded proteins can lead to various diseases, including neurodegenerative disorders like Alzheimer's and Parkinson's. Therefore, a deeper understanding of protein folding is vital for understanding disease mechanisms and developing therapeutic strategies. This study introduces the Stochastic Landscape Classification (SLC), an innovative, automated, nonlearning algorithm that quantitatively analyzes protein folding dynamics. Focusing on collective variables (CVs) - low-dimensional representations of complex dynamical systems like molecular dynamics (MD) of macromolecules - the SLC approach segments the CVs into distinct macrostates, revealing the protein folding pathway explored by MD simulations. The segmentation is achieved by analyzing changes in CV trends and clustering these segments using a standard density-based spatial clustering of applications with noise (DBSCAN) scheme. Applied to the MD-based CV trajectories of Chignolin and Trp-Cage proteins, the SLC demonstrates apposite accuracy, validated by comparing standard classification metrics against ground-truth data. These metrics affirm the efficacy of the SLC in capturing intricate protein dynamics and offer a method to evaluate and select the most informative CVs. The practical application of this technique lies in its ability to provide a detailed, quantitative description of protein folding processes, with significant implications for understanding and manipulating protein behavior in industrial and pharmaceutical contexts.
PubMed: 38924770
DOI: 10.1021/acs.jctc.4c00464 -
Protein Science : a Publication of the... Jul 2024Conserved tryptophan residues are critical for the structure and the stability of β/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are...
Conserved tryptophan residues are critical for the structure and the stability of β/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in β/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of β/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human βB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of βB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of βB2-crystallin vary over time. By using SAXS, we found that the dimer of βB2-crystallin in solution resembled the lattice βB1-crystallin dimer (face-en-face), whereas the tetramer of βB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of βB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of βB2-crystallin in lens.
Topics: Humans; Protein Folding; Tryptophan; beta-Crystallin B Chain; Mutation; Protein Multimerization; Protein Stability; Hydrophobic and Hydrophilic Interactions; Amino Acid Substitution
PubMed: 38924206
DOI: 10.1002/pro.5092 -
The FEBS Journal Jun 2024Especially in higher eukaryotes, the N termini of proteins are subject to enzymatic modifications, with the acetylation of the alpha-amino group of nascent polypeptides... (Review)
Review
Especially in higher eukaryotes, the N termini of proteins are subject to enzymatic modifications, with the acetylation of the alpha-amino group of nascent polypeptides being a prominent one. In recent years, the specificities and substrates of the enzymes responsible for this modification, the Nα-terminal acetyltransferases, have been mapped in several proteomic studies. Aberrant expression of, and mutations in these enzymes were found to be associated with several human diseases, explaining the growing interest in protein Nα-terminal acetylation. With some enzymes, such as the Nα-terminal acetyltransferase A complex having thousands of possible substrates, researchers are now trying to decipher the functional outcome of Nα-terminal protein acetylation. In this review, we zoom in on one possible functional consequence of Nα-terminal protein acetylation; its effect on protein folding. Using selected examples of proteins associated with human diseases such as alpha-synuclein and huntingtin, here, we discuss the sometimes contradictory findings of the effects of Nα-terminal protein acetylation on protein (mis)folding and aggregation.
PubMed: 38923676
DOI: 10.1111/febs.17209 -
Protein Science : a Publication of the... Jul 2024Although in silico folding based on coevolving residue constraints in the deep-learning era has transformed protein structure prediction, the contributions of coevolving...
Although in silico folding based on coevolving residue constraints in the deep-learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well-known histone reader with distinct subtypes containing subtype-specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model-based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.
Topics: Protein Folding; Models, Molecular; Protein Stability; Proteolysis; Humans
PubMed: 38923615
DOI: 10.1002/pro.5065 -
Animal Science Journal = Nihon Chikusan... 2024We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS....
We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS. We procured keratin layer and smooth muscle samples from Holsteins with TAS ranging from 1 to 4, as well as from crossbred heifers (Japanese Black male and Holstein female) with TAS of 1. Teats with a TAS of 4 demonstrated increased total collagen content, higher amounts of type I collagen (the harder, thicker variant), and reduced amounts of type III collagen (the softer, thinner variant) compared with teats with lower TAS. Teats with TAS of 3 and 4 exhibited evidence of damaged collagen in smooth muscle layers compared with teats with TAS of 1. Additionally, we identified 47-kDa heat shock protein-positive fibroblasts in the smooth muscles of teats with TAS of 3 and 4. Therefore, the smooth muscle of teats with a TAS of 4 exhibited increased amounts of denatured collagen in comparison to teats with lower TAS.
Topics: Animals; Cattle; Female; Muscle, Smooth; Collagen; Keratins; Mammary Glands, Animal; Protein Denaturation; Male; Collagen Type I; Fibroblasts; Collagen Type III
PubMed: 38923230
DOI: 10.1111/asj.13969