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To manipulate the protein concentration at a certain functional state through chemical stabilizers is crucial for protein-related studies. It not only plays a key role in protein structure analysis and protein folding kinetics, but also affects protein functionality to a large extent and thus has wide applications in medicine, food industry, etc. However, due to concerns about side effects or financial costs of stabilizers, identifying optimal strategies for enhancing protein stability with a minimal amount of stabilizers is of great importance. Here, we prove that either for the fixed terminal time (including both finite and infinite cases) or for the free one, the optimal control strategy for stabilizing the folding intermediates with a linear strategy for stabilizer addition belongs to the class of bang–bang controls. The corresponding optimal switching time is derived analytically, whose phase diagram with respect to several key parameters is explored in detail. The bang–bang control will be broken when nonlinear strategies for stabilizer addition are adopted. Moreover, the above theory is applied to the stabilization of erythropoietin by ten different kinds of chemicals, providing theoretical guidance for the selection and rational usage of stabilizers. Our current study on optimal strategies for protein stabilizers not only offers deep insights into the general picture of protein folding kinetics but also provides valuable theoretical guidance on treatments for protein-related diseases in medicine. [ABSTRACT FROM AUTHOR]

Parallel tempering molecular dynamics simulation, also known as temperature replica exchange simulation, is a popular enhanced sampling method used to study biomolecular systems. This method makes it possible to calculate the free energy differences between states of the system for a series of temperatures. We developed a method to easily calculate the errors (standard errors or confidence intervals) of these predictions using a modified version of our recently introduced JumpCount method. The number of transitions between states (e.g., protein folding events) is counted for each temperature. This number of transitions, together with the temperature, fully determines the value of the standard error or the confidence interval of the free energy difference. We also address the issue of convergence in the situation where all replicas start from one state by developing an estimator of the equilibrium constant from simulations that are not fully equilibrated. The prerequisite of the method is the Markovianity of the process studied. [ABSTRACT FROM AUTHOR]

Within the confines of a densely populated cell nucleus, chromatin undergoes intricate folding, forming loops, domains, and compartments under the governance of topological constraints and phase separation. This coordinated process inevitably introduces interference between different folding strategies. In this study, we model interphase chromatins as block copolymers with hetero-hierarchical loops within a confined system. Employing dissipative particle dynamics simulations and scaling analysis, we aim to explain how the structure and distribution of loop domains modulate the microphase separation of chromatins. Our results highlight the correlation between the microphase separation of the copolymer and the length, heterogeneity, and hierarchically nested levels of the loop domains. This correlation arises from steric repulsion intrinsic to loop domains. The steric repulsion induces variations in chain stiffness (including local orientation correlations and the persistence length), thereby influencing the degree of phase separation. Through simulations of block copolymers with distinct groups of hetero-hierarchical loop anchors, we successfully reproduce changes in phase separation across diverse cell lines, under fixed interaction parameters. These findings, in qualitative alignment with Hi-C data, suggest that the variations of loop constraints alone possess the capacity to regulate higher-order structures and the gene expressions of interphase chromatins. [ABSTRACT FROM AUTHOR]

We investigate the "roughness" of the energy landscape of a system that diffuses in a heterogeneous medium with a random position-dependent friction coefficient α(x). This random friction acting on the system stems from spatial inhomogeneity in the surrounding medium and is modeled using the generalized Caldira–Leggett model. For a weakly disordered medium exhibiting a Gaussian random diffusivity D(x) = kBT/α(x) characterized by its average value ⟨D(x)⟩ and a pair-correlation function ⟨D(x1)D(x2)⟩, we find that the renormalized intrinsic diffusion coefficient is lower than the average one due to the fluctuations in diffusivity. The induced weak internal friction leads to increased roughness in the energy landscape. When applying this idea to diffusive motion in liquid water, the dissociation energy for a hydrogen bond gradually approaches experimental findings as fluctuation parameters increase. Conversely, for a strongly disordered medium (i.e., ultrafast-folding proteins), the energy landscape ranges from a few to a few kcal/mol, depending on the strength of the disorder. By fitting protein folding dynamics to the escape process from a metastable potential, the decreased escape rate conceptualizes the role of strong internal friction. Studying the energy landscape in complex systems is helpful because it has implications for the dynamics of biological, soft, and active matter systems. [ABSTRACT FROM AUTHOR]

Deep eutectic solvents (DESs) emerged as potential alternative solvent media in multiple areas, including biomolecular (cryo)preservation. Herein, we studied the stability of a small protein (ubiquitin) in water and a betaine-glycerol-water (B:G:W) (1:2:ζ; ζ = 0, 1, 2, 5, 10) DES, through molecular dynamics. An AMBER-based model that accurately describes the density and shear viscosity of the DES is proposed. We find that water molecules are largely trapped in the solvent, precluding the formation of a full hydration layer, seemingly opposite to osmolytes' preferential exclusion/preferential hydration mechanism. Although the protein is stable in the DES, structural fluctuations are largely suppressed and only recovered upon sufficient hydration. This is explained by a solvent-slaving mechanism where β-fluctuations are key, with the non-monotonic hydration of some amino acids with the water content providing an explanation to the non-monotonic folding of some proteins in aqueous DESs. A major thermal stability enhancement in the DES is also observed, caused by a similar slowdown of the backbone torsional dynamics. Our results support a kinetic stabilization of the protein in the DES, whereas a possible thermodynamic stabilization does not follow a preferential hydration or water entrapment mechanism. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii , a unicellular green alga, is a prominent model for green biotechnology and for studying organelles' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii , including the 5' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae. • Successful establishment of an RNA-based transient expression system in Chlamydomonas. • Function of 5' UTR on gene expression occurs at the post-transcriptional level in Chlamydomonas. • Analysis of the localization, function, and interactions of proteins can be completed within one week. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated BamA, the central component of the β-barrel assembly machinery (BAM), as an important therapeutic target to combat infections caused by Acinetobacter baumannii and other Gram-negative pathogens. Homology modeling indicates BamA in A. baumannii consists of five polypeptide transport-associated (POTRA) domains and a β-barrel membrane domain. We characterized the POTRA domains of BamA from A. baumannii in solution using size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) analysis and determined crystal structures in two conformational states that are drastically different than those previously observed in BamA from other bacteria, indicating that the POTRA domains are even more conformationally dynamic than has been observed previously. Molecular dynamics simulations of the POTRA domains from A. baumannii and Escherichia coli allowed us to identify key structural features that contribute to the observed novel states. Together, these studies expand on our current understanding of the conformational plasticity within BamA across differing bacterial species. [Display omitted] • The POTRA domains of BamA are known to be flexible • Crystal structures of A. baumannii BamA POTRA domains reveal novel conformations • MD simulations and SAXS data are consistent with the crystal structures • These new conformations may explain how Bam components become surface exposed In A. baumannii , BamA has an N-terminal periplasmic domain consisting of five tandem POTRA domains. Overly Cottom et al. determined crystal structures of AbBamA POTRAs 1–4, revealing new conformations that are rotated by ∼180°, indicating even more conformational plasticity than has been observed previously and offering a possible explanation for surface exposure of accessory proteins. [ABSTRACT FROM AUTHOR]

Malformations of the brain are common and vary in severity, from negligible to potentially fatal. Their causes have not been fully elucidated. Here, we report pathogenic variants in the core protein-folding machinery TRiC/CCT in individuals with brain malformations, intellectual disability, and seizures. The chaperonin TRiC is an obligate hetero-oligomer, and we identify variants in seven of its eight subunits, all of which impair function or assembly through different mechanisms. Transcriptome and proteome analyses of patient-derived fibroblasts demonstrate the various consequences of TRiC impairment. The results reveal an unexpected and potentially widespread role for protein folding in the development of the central nervous system and define a disease spectrum of “TRiCopathies.” [ABSTRACT FROM AUTHOR]

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70‐HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a‐responsive‐responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability. Summary statement: This work describes that HOP cochaperones play a major role in thermomorphogenesis by modulating the stability of the HSFA1a in Arabidopsis. [ABSTRACT FROM AUTHOR]

The biological denitrification process is crucial for removing nitrogen and reducing nitrous oxide (N2O) emissions. To investigate the mechanism of action of key enzymes in the denitrification process, it is necessary to analyze the protein structure, active site, and conformational changes of denitrifying enzymes. X-ray crystallography, nuclear magnetic resonance (NMR), and cryogenic electron microscopy (Cryo-EM), as well as spectroscopic analyses such as Raman resonance and UV-vis, have been used to study the structure of denitrifying enzymes. However, practical application is limited by protein folding and assembly, as well as issues of purity, concentration, and homogeneity. This review summarizes the biochemical properties of the four denitrifying enzymes and typical purification methods. Additionally, this review emphasizes important factors to consider during protein expression, crude extract, and chromatographic purification. It also provides insight into the future development of protein purification methods for denitrifying enzymes. This review aims to bridge the gap between the complex biochemical process of denitrifying enzymes and the technical intricacies of protein purification, with the ultimate goal of reducing N2O emissions in ecosystems. [ABSTRACT FROM AUTHOR]

This work examines the thermodynamics of model biomolecular interactions using a governing equation that accounts for the participation of bulk water in the equilibria. In the first example, the binding affinities of two DNA duplexes, one of nine and one of 10 base pairs in length, are measured and characterized by isothermal titration calorimetry (ITC) as a function of concentration. The results indicate that the change in solvation free energy that accompanies duplex formation (ΔGS) is large and unfavorable. The duplex with the larger number of G:C pairings yields the largest change in solvation free energy, ΔGS = +460 kcal·mol−1per base pair at 25 °C. A van't Hoff analysis of the data is complicated by the varying degree of intramolecular base stacking within each DNA strand as a function of temperature. A modeling study reveals how the solvation free energy alters the output of a typical ITC experiment and leads to a good, though misleading, fit to the classical equilibrium equation. The same thermodynamic framework is applied to a model protein–ligand interaction, the binding of ribonuclease A with the nucleotide inhibitor 3′‐UMP, and to a conformational equilibrium, the change in tertiary structure of α‐lactalbumin in molar guanidinium chloride solutions. The ribonuclease study yields a value of ΔGS = +160 kcal·mol−1, whereas the folding equilibrium yields ΔGS ≈ 0, an apparent characteristic of hydrophobic interactions. These examples provide insight on the role of solvation energy in binding equilibria and suggest a pivot in the fundamental application of thermodynamics to solution chemistry. [ABSTRACT FROM AUTHOR]

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of CG4511 as Drosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5' UTR of the PhLP3 gene exhibited significant downregulation of PhLP3 expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in PhLP3 restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species. [ABSTRACT FROM AUTHOR]

The formation of inclusion bodies (IBs) in microbial cell factories is a very common process occurring during recombinant protein production. Different protocols have been developed for the extraction of soluble proteins from IBs using several strategies, ranging from the use of harsh denaturing and high concentrations of chaotropic agents and reducing agents to the use of mild protocols based on the use of non-denaturing detergents. However, in recent years, the biological vision of IBs has changed and research studies have demonstrated that these protein aggregates contain biologically active and properly folded recombinant proteins. This drives us to redefine the methodologies currently used to obtain soluble protein using IB as a protein source. Hence, we propose the extraction of IB protein via the simple spontaneous solubilization of IB as a strategy broadly applicable to all kinds of recombinant proteins without the negative effects of detergents and chaotropic agents on final biological activity. We prove the wide applicability of spontaneous solubilization processes to different types of IBs and that protocols can be easily customized for each protein in terms of timing and incubation temperature by monitoring the protein activity of the solubilized fraction. [ABSTRACT FROM AUTHOR]

The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP's function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP's functions during infection by the Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies. Author summary: The endoplasmic reticulum (ER) chaperone protein BiP (HSPA5) plays a central role in protein folding and maintaining homeostasis within the ER. Under certain conditions, such as cancer and viral infections, BiP is dysregulated to support cell survival or viral replication. In this study, we investigated the regulation and requirement of BiP during infection by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic herpesvirus linked to cancers in immunocompromised individuals. Our findings demonstrate that BiP is significantly upregulated in KSHV-infected cells, even when the expression of most other cellular genes is suppressed. Notably, the function of BiP is essential for KSHV replication and the survival of KSHV-infected cells. This reliance on BiP is not unique to KSHV; it is also required for the replication of other double-stranded DNA (dsDNA) viruses, including Herpes simplex virus I, Human Cytomegalovirus, and Vaccinia Virus. These findings underscore the critical role of BiP in dsDNA viral infections, positioning this chaperone as a promising target for the development of broad-spectrum antiviral therapies and potential treatment strategies for KSHV-associated malignancies. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimer's disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions. This study introduces an in vivo protein footprinting method, revealing structural changes in proteome during progressing AD. It demonstrates these changes occur before expression alterations, advancing understanding of protein misfolding mechanisms. [ABSTRACT FROM AUTHOR]

Expanding the protein fold space beyond linear chains is of fundamental significance, yet remains largely unexplored. Herein, we report the creation of seven topological isoforms (i.e., linear, cyclic, knot, lasso, pseudorotaxane, and catenane) from a single protein fold precursor by rewiring the connectivity of secondary structure elements of the SpyTag-SpyCatcher complex and mutating the reactive residue on SpyTag to abolish the isopeptide bonding. These topological isoforms can be directly expressed in cells. Their topologies were confirmed by combined techniques of proteolytic digestion, fluorescence correlation spectroscopy (FCS), size-exclusion chromatography (SEC), and topological transformation. To study the effects of topology on their structures and properties, their biophysical properties were characterized by differential scanning calorimetry (DSC), heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC-NMR), and circular dichroism (CD) spectroscopy. Molecular dynamics (MD) simulations were further performed to reveal the atomic details of structural changes upon unfolding. Both experimental and simulation results suggest that they share a similar, well-folded hydrophobic core but exhibit distinct folding/unfolding dynamic behaviors. These results shed light onto the folding landscape of topological isoforms derived from the same protein fold. As a model system, this work improves our understanding of protein structure and dynamics beyond linear chains and suggests that protein folds are highly amenable to topological variation. [ABSTRACT FROM AUTHOR]

π–π stacking interactions are versatile because they are involved in many processes, such as protein folding, DNA stacking, and drug recognition. However, from the point of view of crystal engineering, there is an incipient knowledge of its exploitation. A comparison of these interactions with hydrogen bonds shows a huge difference in their employment as a reliable non-covalent interaction. And different reasons can be listed to explain why hydrogen bonding can be considered a more robust interaction than π−π stacking. For instance, hydrogen bonds encompass a wide energy range (25–40 kJ mol−1). From this, these interactions can be classified as strong, moderate, and weak. Hence, the first two can be considered highly to moderately directional to be exploited in crystal engineering. This aspect is relevant for them to be used in a relatively reliably way in this area of supramolecular chemistry. On the other hand, in the case of π−π stacking, the energy range is 0–10 kJ mol−1, thus implying that hydrogen bonds or any other energetically more robust contact would predominate in the competition for establishing packing interactions in a given arrangement. In this sense, if stacking is pretended to be exploited from the point of view of crystal engineering, one of the points that must be ensured is that this interaction will be the one energetically predominant. However, although there are other factors to consider, it seems that energetics is the dominant one. In this line, our research group has obtained and studied many single-crystalline structures of coordination and organometallic compounds containing fluorinated thiolates. This being particularly true in the case of the thiolate 2,3,5,6-S(C6F4H-4) bound to different metals, where it has been observed that they preferentially tend to establish πF–πF stacking interactions, results that have been reported in several papers. Thus, from this perspective, we have explored, using ConQuest (CCDC) a number of structures to observe how feasible is to find stacking in coordination and organometallic compounds containing the thiolate 2,3,5,6-S(C6F4H-4). [ABSTRACT FROM AUTHOR]

The function of a biomacromolecule is not only determined by its three-dimensional structure but also by its electronic state. Quantum chemical calculations are promising non-empirical methods available for determining the electronic state of a given structure. In this study, we used the fragment molecular orbital (FMO) method, which applies to biopolymers such as proteins, to provide physicochemical property values on representative structures in the SCOP2 database of protein families, a subset of the Protein Data Bank. Our dataset was constructed by over 5,000 protein structures, including over 200 million inter-fragment interaction energies (IFIEs) and their energy components obtained by pair interaction energy decomposition analysis (PIEDA) using FMO-MP2/6-31 G*. Moreover, three basis sets, 6-31 G*, 6-31 G**, and cc-pVDZ, were used for the FMO calculations of each structure, making it possible to compare the energies obtained with different basis functions for the same fragment pair. The total data size is approximately 6.7 GB. Our dataset will be useful for functional analyses and machine learning based on the physicochemical property values of proteins. [ABSTRACT FROM AUTHOR]

Klotho is an anti-aging protein whose deletion significantly reduces lifespan in mice, while its over-expression increases lifespan. Klotho is a type-I transmembrane protein that is N-glycosylated at eight positions within its ectodomain. Our study demonstrates that N-glycosylation or mutation at position N614, but not at N161, N285, or N346 in mouse Klotho, is critically involved in the transport of Klotho out of the endoplasmic reticulum (ER). Consequently, while wild-type Klotho-EGFP as well as the N-glycosylation mutants N161Q, N285Q, and N346Q were present at the plasma membrane (PM), only small amounts of the N614Q Klotho-EGFP were present at the PM, with most of the protein accumulating in the ER. Protein interactome analysis of Klotho-EGFP N614Q revealed increased interactions with proteasome-related proteins and proteins involved in ER protein processing, like heat shock proteins and protein disulfide isomerases, indicative of impaired protein folding. Co-immunoprecipitation experiments confirmed the interaction of Klotho-EGFP N614Q with ER chaperons. Interestingly, despite the low amounts of Klotho-EGFP N614Q at the PM, it efficiently induced FGF receptor-mediated ERK activation in the presence of FGF23, highlighting its efficacy in triggering downstream signaling, even in limited quantities at the PM. [ABSTRACT FROM AUTHOR]

Mpox, caused by the Mpox virus (MPXV), emerged globally in 2022 with the Clade IIb strain, presenting a critical public health challenge. While MPXV is primarily characterized by fever and rash, gastrointestinal (GI) complications, such as diarrhea and proctitis, have also been observed. This study is a reanalysis of GSE219036 without own data and focuses on the impact of MPXV infection on the colon, using human-induced pluripotent stem cell-derived colon organoids as a model. We applied a tailored statistical framework for RNA-seq data, Generalized Linear Models with Quasi-Likelihood F-tests and Relaxed Magnitude–Altitude Scoring (GLMQL-RMAS), to identify differentially expressed genes (DEGs) across MPXV clades: MPXV I (Zr-599 Congo Basin), MPXV IIa (Liberia), and MPXV IIb (2022 MPXV). Through a novel methodology called Cross-RMAS, we ranked genes by integrating statistical significance and biological relevance across all clades. Machine learning analysis using the genes identified by Cross-RMAS, demonstrated 100% accuracy in differentiating between the different MPXV strains and mock samples. Furthermore, our findings reveal that MPXV Clade I induces the most extensive alterations in gene expression, with significant upregulation of stress response genes, such as HSPA6 and FOS, and downregulation of genes involved in cytoskeletal organization and vesicular trafficking, such as PSAP and CFL1. In contrast, Clade IIb shows the least impact on gene expression. Through Gene Ontology (GO) analysis, we identified pathways involved in protein folding, immune response, and epithelial integrity that are disrupted in infected cells, suggesting mechanisms by which MPXV may contribute to GI symptoms. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD), a spectrum of liver conditions characterized by fat accumulation without excessive alcohol consumption, represents a significant global health burden. The intricate molecular landscape underlying NAFLD pathogenesis involves lipid handling, inflammation, oxidative stress, and mitochondrial dysfunction, with endoplasmic reticulum (ER) stress emerging as a key contributor. ER stress triggers the unfolded protein response (UPR), impacting hepatic steatosis in NAFLD and contributing to inflammation, fibrosis, and progression to NASH and eventually hepatocellular carcinoma (HCC). Heat shock proteins (HSPs), including small HSPs such as HSP20 and HSP27, HSP60, HSP70, GRP78, and HSP90, are integral to cellular stress responses. They aid in protein folding, prevent aggregation, and facilitate degradation, thus mitigating cellular damage under stress conditions. In NAFLD, aberrant HSP expression and function contribute to disease pathogenesis. Understanding the specific roles of HSP subtypes in NAFLD offers insights into potential therapeutic interventions. This review discusses the involvement of HSPs in NAFLD pathophysiology and highlights their therapeutic potential. By elucidating the molecular mechanisms underlying HSP-mediated protection in NAFLD, this article aims to pave the way for the development of targeted therapies for this prevalent liver disorder. [ABSTRACT FROM AUTHOR]

Elucidating the role of molecular chaperones in extremely thermophilic archaea, including the gamma prefoldin (γPFD) in the deep‐sea methanogen Methanocaldococcus jannaschii, is integral to understanding microbial adaptation to hot environments. This study focuses on genetically engineered knock‐out and overexpression strains to evaluate the importance of γPFD in the growth and thermal tolerance of M. jannaschii. An in‐depth analysis of cell growth, morphology and transcriptional responses to heat stress revealed that although the gene encoding γPFD is substantially upregulated in response to heat shock, the γPFD is not indispensable for high‐temperature survival. Instead, its absence in the knock‐out strain is compensated for by the upregulation of several proteolytic proteins in the absence of heat shock, nearly matching the corresponding transcription profile of selected transcripts for proteins involved in protein synthesis and folding in the wild‐type strain following heat shock, using quantitative reverse‐transcription PCR (RT‐qPCR). These findings bridge environmental adaptation with molecular biology, underscoring the versatility of extremophiles and providing a deeper mechanistic understanding of how they cope with stress. [ABSTRACT FROM AUTHOR]

Significance: Oxidative folding within the endoplasmic reticulum (ER) introduces disulfide bonds into nascent polypeptides, ensuring proteins' stability and proper functioning. Consequently, this process is critical for maintaining proteome integrity and overall health. The productive folding of thousands of secretory proteins requires stringent quality control measures, such as the unfolded protein response (UPR) and ER-Associated Degradation (ERAD), which contribute significantly to maintaining ER homeostasis. ER-localized protein disulfide isomerases (PDIs) play an essential role in each of these processes, thereby contributing to various aspects of ER homeostasis, including maintaining redox balance, proper protein folding, and signaling from the ER to the nucleus. Recent Advances: Over the years, there have been increasing reports of the (re)localization of PDI family members and other ER-localized proteins to various compartments. A prime example is the anterior gradient (AGR) family of PDI proteins, which have been reported to relocate to the cytosol or the extracellular environment, acquiring gain of functions that intersect with various cellular signaling pathways. Critical Issues: Here, we summarize the functions of PDIs and their gain or loss of functions in non-ER locations. We will focus on the activity, localization, and function of the AGR proteins: AGR1, AGR2, and AGR3. Future Directions: Targeting PDIs in general and AGRs in particular is a promising strategy in different human diseases. Thus, there is a need for innovative strategies and tools aimed at targeting PDIs; those strategies should integrate the specific localization and newly acquired functions of these PDIs rather than solely focusing on their canonical roles. [ABSTRACT FROM AUTHOR]

Graphical models are pivotal in studying the conditional independence structure of a set of random variables. Circular variables, arising in several contexts and fields, are characterized by periodicity. Models for studying the dependence/independence structure of circular variables are under-explored but of increasing interest. This paper delves into two multivariate circular distributions, the Wrapped Normal and the Inverse Stereographic Normal distribution as undirected graphical models. For each of these distributions, we study their key properties with respect to conditional independence and introduce specific classes of graphical models. The usefulness of the proposal is shown by modelling the conditional independence among dihedral angles that play a critical role in defining the three-dimensional structure and functionality of proteins. This can provide valuable insights, for instance, into the multiform protein folding understanding. [ABSTRACT FROM AUTHOR]

Genome instability relies on preserving the chromatin structure, with any histone imbalances threating DNA integrity. Histone synthesis occurs in the cytoplasm, followed by a maturation process before their nuclear translocation. This maturation involves protein folding and the establishment of post-translational modifications. Disruptions in this pathway hinder chromatin assembly and contribute to genome instability. JMJD1B, a histone demethylase, not only regulates gene expression but also ensures a proper supply of histones H3 and H4 for the chromatin assembly. Reduced JMJD1B levels lead to the cytoplasmic accumulation of histones, causing defects in the chromatin assembly and resulting in DNA damage. To investigate the role of JMJD1B in regulating genome stability and the malignancy of melanoma tumors, we used a JMJD1B/KDM3B knockout in B16F10 mouse melanoma cells to perform tumorigenic and genome instability assays. Additionally, we analyzed the transcriptomic data of human cutaneous melanoma tumors. Our results show the enhanced tumorigenic properties of JMJD1B knockout melanoma cells both in vitro and in vivo. The γH2AX staining, Micrococcal Nuclease sensitivity, and comet assays demonstrated increased DNA damage and genome instability. The JMJD1B expression in human melanoma tumors correlates with a lower mutational burden and fewer oncogenic driver mutations. Our findings highlight JMJD1B's role in maintaining genome integrity by ensuring a proper histone supply to the nucleus, expanding its function beyond gene expression regulation. JMJD1B emerges as a crucial player in preserving genome stability and the development of melanoma, with a potential role as a safeguard against oncogenic mutations. [ABSTRACT FROM AUTHOR]

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3. [ABSTRACT FROM AUTHOR]

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP‐dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP‐dependent manner through charge‐driven interactions. Furthermore, the Hsp60β homo‐oligomeric complex can protect the archaeal and eukaryotic membrane from stress‐induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC‐type cooperativity is nested within KNF‐type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality. [ABSTRACT FROM AUTHOR]

SUMMARY: Structural prediction by artificial intelligence can be powerful new instruments to discover novel protein–protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re‐analyse our in silico screen for novel pathogen‐secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations caused by different platforms, sequence depth and computing times. We hope these experiences will support similar interactomic screens by the research community. [ABSTRACT FROM AUTHOR]

GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroELMEL, in which the 307‒309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroELMEL-mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent. [Display omitted] [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The strategy of removable glycosylation modification was used to overcome the low‐efficiency problem encountered in the chemical synthesis of the mirror‐image D‐version of the immunoglobulin (Ig)‐like domain of tropomyosin receptor kinase A (DlgCTrkA), a protein molecule needed for mirror‐image screening of D‐peptide ligands targeting this cell membrane receptor. It was found that O‐linked‐β‐N‐acetyl‐D‐glucosamine (O‐GlcNAc) modification at DSer312, or DSer320 can significantly improve the efficiency of DlgCTrkA synthesis and folding, while O‐GlcNAc modification at DSer330 showed barely any improvement. This study provides a new example demonstrating the power of the removable glycosylation modification strategy in the chemical synthesis and folding of difficult‐to‐obtain proteins. It also presents evidence that removable glycosylation modification at different sites would significantly affect the efficiency of protein folding promoted by this strategy. [ABSTRACT FROM AUTHOR]

The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of soluble proteins into the compartment, where they are retained through oxidative folding. Additionally, the disulfide relay enhances the stability of many proteins by forming disulfide bonds. In this review, we describe the mitochondrial disulfide relay in human cells, its components, and their coordinated collaboration in mechanistic detail. We also discuss the human pathologies associated with defects in this machinery and its protein substrates, providing a comprehensive overview of its biological importance and implications for health. [ABSTRACT FROM AUTHOR]

The kinetostatic compliance method (KCM) models protein molecules as nanomechanisms consisting of numerous rigid peptide plane linkages. These linkages articulate with respect to each other through changes in the molecule dihedral angles, resulting in a kinematic mechanism with hyper degrees of freedom. Within the KCM framework, nonlinear interatomic forces drive protein folding by guiding the molecule's dihedral angle vector towards its lowest energy state in a kinetostatic manner. This paper proposes a numerical integrator that is well suited to KCM-based protein folding and overcomes the limitations of traditional explicit Euler methods with fixed step size. Our proposed integration scheme is based on pseudo-transient continuation with an adaptive step size updating rule that can efficiently compute protein folding pathways, namely, the transient three-dimensional configurations of protein molecules during folding. Numerical simulations utilizing the KCM approach on protein backbones confirm the effectiveness of the proposed integrator. [ABSTRACT FROM AUTHOR]

The genome editing technology based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein- 9 (CRISPR/Cas9), is revolutionizing research, particularly in the context of human neurodegenerative disorders. This review examines recent advancements in CRISPR/Cas9 and its potential to address the protein misfolding mechanisms underlying these diseases. Proteins, the fundamental units of life, can misfold due to various changes, resulting in aggregation and contributing to devastating illnesses such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and Huntington's disease. Understanding the pathology of these disorders and the methods used for their detection is vital for developing effective treatments. CRISPR/Cas9 offers a powerful tool for combating neurodegenerative disorders at the molecular level. Its groundbreaking gene-editing capabilities are advancing preclinical and animal studies, paving the way for potential human trials and innovative therapeutic strategies. This review explores the complex challenge of protein misfolding and highlights how CRISPR technology could provide a crucial breakthrough in the fight against neurodegenerative disorders. It offers a synthesis of CRISPR advancements for neurodegenerative disorders. However, it is essential to be aware of the review's limitations, including potential selection bias, the risk of oversimplification, and possible obsolescence in rapidly changing research fields. Despite these considerations, the transformative promise of CRISPR in understanding and potentially treating neurodegenerative diseases warrants continued research and thorough analysis. [ABSTRACT FROM AUTHOR]

Fold‐switching enables metamorphic proteins to reversibly interconvert between two highly dissimilar native states to regulate their protein functions. While about 100 proteins have been identified to undergo fold‐switching, unveiling the key residues behind this mechanism for each protein remains challenging. Reasoning that fold‐switching in proteins is driven by dynamic changes in local energetic frustration, we combined fold‐switching simulations generated using simplified structure‐based models with frustration analysis to identify key residues involved in this process based on the change in the density of minimally frustrated contacts during refolding. Using this approach to analyze the fold‐switch of the bacterial transcription factor RfaH, we identified 20 residues that significantly change their frustration during its fold‐switch, some of which have been experimentally and computationally reported in previous works. Our approach, which we developed as an additional module for the FrustratometeR package, highlights the role of local frustration dynamics in protein fold‐switching and offers a robust tool to enhance our understanding of other proteins with significant conformational shifts. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The strategy of removable glycosylation modification was used to overcome the low‐efficiency problem encountered in the chemical synthesis of the mirror‐image D‐version of the immunoglobulin (Ig)‐like domain of tropomyosin receptor kinase A (DlgCTrkA), a protein molecule needed for mirror‐image screening of D‐peptide ligands targeting this cell membrane receptor. It was found that O‐linked‐β‐N‐acetyl‐D‐glucosamine (O‐GlcNAc) modification at DSer312, or DSer320 can significantly improve the efficiency of DlgCTrkA synthesis and folding, while O‐GlcNAc modification at DSer330 showed barely any improvement. This study provides a new example demonstrating the power of the removable glycosylation modification strategy in the chemical synthesis and folding of difficult‐to‐obtain proteins. It also presents evidence that removable glycosylation modification at different sites would significantly affect the efficiency of protein folding promoted by this strategy. [ABSTRACT FROM AUTHOR]

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils. In this paper, the authors revealed the distinct folding motifs for irreversible and reversible amyloids derived from a single protein, by combining multiple mesoscopic and molecular techniques including AFM, cryo-EM and solid-state NMR. [ABSTRACT FROM AUTHOR]

Iminosugars, a class of polyhydroxylated cyclic alkaloids with intriguing properties, hold promising therapeutic potentials against a broad spectrum of enveloped viruses, including DENV, HCV, HIV, and influenza viruses. Mechanistically, iminosugars act as the competitive inhibitors of host endoplasmic reticular α-glucosidases I and II to disrupt the proper folding of viral nascent glycoproteins, which thereby exerts antiviral effects. Remarkably, the glycoproteins of many enveloped viruses are significantly more dependent on the calnexin pathway of the protein folding than most host glycoproteins. Therefore, extensive interests and efforts have been devoted to exploit iminosugars as broad-spectrum antiviral agents. This review provides the summary and insights into the recent advancements in the development of novel iminosugars as effective and selective antiviral agents against a variety of enveloped viruses, as well as the understandings of their antiviral mechanisms. [ABSTRACT FROM AUTHOR]

Germinating seedlings emit light in the visible range spontaneously, and these emissions are related to metabolism and reactive-oxygen species (ROS) processes. Several series of germination tests had such biological autoluminescence (BAL) recorded in controlled conditions, fostering applications for the non-invasive, real-time evaluation of a seedling's germinability and vigor when submitted to chemical and/or physical perturbations. However, long-term analysis of the BAL time-series of control samples conducted in different locations around the globe reveals that their BAL signals (and thus their metabolism) appear to follow the local gravimetric tide (g-tide) time profile. This indicates that the small daily and monthly variations in gravity acceleration due to the relative positioning of the Sun and Moon with respect to the Earth's surface influence these signals. The gravimetric tide is a natural phenomenon that affects all things, fluid or solid, and is an uncontrollable variable in normal laboratory settings on Earth's crust. All things on Earth have evolved under these pervasive cycles, with periods ranging from ~12.2 h up to 28 d, which correspond to the Moon cycle. Tide-like cycles occur in living beings of different types, from the simplest bacteria to the most complex human beings, and we speculate that water availability at the molecular level could be a significant physical factor in modulating bio-activity by enabling protein folding and all metabolic pathways that require a synchronized organization to adapt to external environmental conditions. In this study, we summarize published results of a seedling's BAL with cycle patterns resembling the g-tide in Limeira/BR, Prague/CZ, Leiden/NL, and Hamamatsu/JP and discuss the possible implications of this phenomenon for chronobiological studies. [ABSTRACT FROM AUTHOR]

The mechanical properties of proteins/peptides play an essential role in their functionalities and implications, as well as their structure and dynamic properties. Understanding mechanical properties is pivotal to our knowledge of protein folding and the molecular basis of diverse cellular processes. Herein, we present a computational approach using ab initio quantum mechanical calculations to determine the mechanical properties—such as bulk modulus, shear modulus, Young's modulus, and Poisson's ratio—of a solvated Arg-Gly-Asp (RGD) peptide model. Since this peptide serves as the RGD-directed integrin recognition site and may participate in cellular adhesion, it is considered a promising small peptide for medicinal applications. This successful approach paves the way for investigating larger and more complex biomolecules. [ABSTRACT FROM AUTHOR]

This review offers an in-depth examination of amyotrophic lateral sclerosis (ALS), addressing its epidemiology, pathophysiology, clinical presentation, diagnostic techniques, and current as well as emerging treatments. The purpose is to condense key findings and illustrate the complexity of ALS, which is shaped by both genetic and environmental influences. We reviewed the literature to discuss recent advancements in understanding molecular mechanisms such as protein misfolding, mitochondrial dysfunction, oxidative stress, and axonal transport defects, which are critical for identifying potential therapeutic targets. Significant progress has been made in refining diagnostic criteria and identifying biomarkers, leading to earlier and more precise diagnoses. Although current drug treatments provide some benefits, there is a clear need for more effective therapies. Emerging treatments, such as gene therapy and stem cell therapy, show potential in modifying disease progression and improving the quality of life for ALS patients. The review emphasizes the importance of continued research to address challenges such as disease variability and the limited effectiveness of existing treatments. Future research should concentrate on further exploring the molecular foundations of ALS and developing new therapeutic approaches. The implications for clinical practice include ensuring the accessibility of new treatments and that healthcare systems are equipped to support ongoing research and patient care. [ABSTRACT FROM AUTHOR]

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this “cataract-reversing” phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress–related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates. [ABSTRACT FROM AUTHOR]

Schizophrenia (SCZ) is a severe neuropsychiatric disorder characterized by cognitive, affective, and social dysfunction, resulting in hallucinations, delusions, emotional blunting, and disordered thinking. In recent years, proteomics has been increasingly influential in SCZ research. Glycosylation, a key post-translational modification, can alter neuronal stability and normal signaling in the nervous system by affecting protein folding, stability, and cellular signaling. Recent research evidence suggests that abnormal glycosylation patterns exist in different brain regions in autopsy samples from SCZ patients, and that there are significant differences in various glycosylation modification types and glycosylation modifying enzymes. Therefore, this review explores the mechanisms of aberrant modifications of N-glycosylation, O-glycosylation, glycosyltransferases, and polysialic acid in the brains of SCZ patients, emphasizing their roles in neurotransmitter receptor function, synaptic plasticity, and neural adhesion. Additionally, the effects of antipsychotic drugs on glycosylation processes and the potential for glycosylation-targeted therapies are discussed. By integrating these findings, this review aims to provide a comprehensive perspective to further understand the role of aberrant glycosylation modifications in the pathophysiology of SCZ. [ABSTRACT FROM AUTHOR]

Background: 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. Methods and findings: 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 antirPvQSOX 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. Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization. [ABSTRACT FROM AUTHOR]

Purpose: To explore the plasma proteomic changes of rabbit lung VX2 tumors treated by microwave ablation, and to explore the molecular pathway mechanisms that may be involved. Methods: New Zealand white rabbits were inoculated with VX2 tumor cell suspension in the right lower lung and treated with microwave ablation after 2-3 weeks of tumor formation. Blood was collected at 5 time points (TP1~TP5) before and after ablation by cardiac blood sampling and pre-treated before proteomic analysis. The plasma proteome was analyzed by Data-Independent Acquisition (DIA). Results: Different molecular pathways were activated at different time points:(i) TP1vsTP2: more proteins were down-regulated and enrichment analysis showed that the proteasome pathway was activated. The abnormal protein folding process involved in this pathway is closely related to the process of tumor development. (ii) TP2vsTP3: more proteins were up-regulated although the number of differentially differentiated proteins was lower and enrichment analysis showed that the phagosome pathway was activated. After microwave ablation inactivates tumor cells, it activates the phagosomal pathway for immune clearance of necrotic tumor tissue. (iii) TP3vsTP4: more down-regulated proteins, enrichment analysis showed that cysteine and methionine metabolism pathway was activated. Decreased metabolism of these amino acids suggests that cancer progression may be blocked after microwave ablation therapy. (iv) TP4vsTP5: the number of differential proteins was less and more down-regulated proteins, enrichment analysis showed that glutathione metabolism and metabolism of xenobiotics by cytochrome P450 pathway were activated. The down-regulated proteins in this pathway may suggest that microwave ablation may have reduced resistance to certain chemotherapeutic agents following. Conclusions: In the process of lung cancer treatment by microwave ablation, the changes of proteins on the possible molecular pathways at each time point are related to lung cancer, and not only involve some simple inflammatory reactions, and some of the proteins released by destroying the tumor cells can be used as possible drug binding sites and reduce drug resistance. [ABSTRACT FROM AUTHOR]

The expansion of high-quality, low-cost sequencing has created an enormous opportunity to understand how genetic variants alter cellular behaviour in disease. The high diversity of mutations observed has however drawn a spotlight onto the need for predictive modelling of mutational effects on phenotype from variants of uncertain significance. This is particularly important in the clinic due to the potential value in guiding clinical diagnosis and patient treatment. Recent computational modelling has highlighted the importance of mutation induced protein misfolding as a common mechanism for loss of protein or domain function, aided by developments in methods that make large computational screens tractable. Here we review recent applications of this approach to different genes, and how they have enabled and supported subsequent studies. We further discuss developments in the approach and the role for the approach in light of increasingly high throughput experimental approaches. [ABSTRACT FROM AUTHOR]

This editorial titled "Advances in Computational Intelligence-Based Methods of Structure and Function Prediction of Proteins" explores the significance of proteins in cellular processes and the role of protein sequence in determining protein structure. It discusses the use of computational intelligence-based models and approaches in predicting protein structures, including template-based modeling, ab initio modeling, and AI-aided methods. The document also provides a list of representative predictors for each category. Additionally, it presents research articles on various topics related to protein folding, secondary structure prediction, and protein function prediction. The document is a summary of a special issue of Biomolecules, which includes research articles and reviews on various topics related to protein folding, secondary structure prediction, HtrA protease family, SARS-CoV-2 spike variant complexes, heme distortion, Trp305, enzyme substrate promiscuity, and hepatitis C virus genome. The authors disclose their contributions and funding sources and declare no conflicts of interest. The article includes references to other relevant research articles and resources. It is important to note that MDPI and/or the editor(s) are not responsible for any harm caused by the ideas, methods, instructions, or products mentioned in the content, emphasizing the need for caution and further investigation when considering the information presented. [Extracted from the article]

The solvent-induced interactions (SIIs) between flexible solutes can be separated into two distinct components: the solvation-induced conformational effect and the joint solvation interaction (JSI). The JSI quantifies the thermodynamic effect of the solvent simultaneously accommodating the solutes, generalizing the typical notion of the hydrophobic interaction. We present a formal definition of the JSI within the framework of the mixture expansion, demonstrate that this definition is equivalent to the SII between rigid solutes, and propose a method, partially connected molecular dynamics, which allows one to compute the interaction with existing free energy algorithms. We also compare the JSI to the more natural generalization of the hydrophobic interaction, the indirect solvent-mediated interaction, and argue that JSI is a more useful quantity for studying solute binding thermodynamics. Direct calculation of the JSI may prove useful in developing our understanding of solvent effects in self-assembly, protein aggregation, and protein folding, for which the isolation of the JSI from the conformational component of the SII becomes important due to the intra-species flexibility. [ABSTRACT FROM AUTHOR]

Although unfolded protein response (UPR) is essential for cellular protection, its prolonged activation may induce apoptosis, compromising cellular longevity. The aging process increases the endoplasmic reticulum (ER) stress in skeletal muscle. However, whether combined exercise can prevent age-induced ER stress in skeletal muscle remains unknown. Evidence suggests that ER stress may increase inflammation by counteracting the positive effects of interleukin-10 (IL-10), whereas its administration in cells inhibits ER stress and apoptosis. This study verified the effects of aging and combined exercise on physical performance, ER stress markers, and inflammation in the quadriceps of mice. Moreover, we verified the effects of IL-10 on ER stress markers. C57BL/6 mice were distributed into young (Y, 6 mo old), old sedentary (OS, sedentary, 24 mo old), and old trained group (OT, submitted to short-term combined exercise, 24 mo old). To clarify the role of IL-10 in UPR pathways, knockout mice lacking IL-10 were used. The OS mice presented worse physical performance and higher ER stress-related proteins, such as C/EBP homologous protein (CHOP) and phospho-eukaryotic translation initiation factor 2 alpha (p-eIF2α/eIF2α). The exercise protocol increased muscle strength and IL-10 protein levels in OT while inducing the downregulation of CHOP protein levels compared with OS. Furthermore, mice lacking IL-10 increased BiP, CHOP, and p-eIF2α/eIF2α protein levels, indicating this cytokine can regulate the ER stress response in skeletal muscle. Bioinformatics analysis showed that endurance and resistance training downregulated DNA damage inducible transcript 3 (DDIT3) and XBP1 gene expression in the vastus lateralis of older people, reinforcing our findings. Thus, combined exercise is a potential therapeutic intervention for promoting adjustments in ER stress markers in aged skeletal muscle. NEW & NOTEWORTHY: Aging elevates endoplasmic reticulum (ER) stress in skeletal muscle, potentially heightening inflammation by opposing interleukin-10 (IL-10) effects. This study found that short-term combined exercise boosted strength and IL-10 protein levels while reducing CHOP protein levels in older mice. In addition, IL-10-deficient mice exhibited increased ER stress markers, highlighting IL-10's role in regulating ER stress in skeletal muscle. Consequently, combined exercise emerges as a therapeutic intervention to elevate IL-10 and adjust ER stress markers in aging. [ABSTRACT FROM AUTHOR]

Protein dynamics play a crucial role in biological function, encompassing motions ranging from atomic vibrations to large-scale conformational changes. Recent advancements in experimental techniques, computational methods, and artificial intelligence have revolutionized our understanding of protein dynamics. Nuclear magnetic resonance spectroscopy provides atomic-resolution insights, while molecular dynamics simulations offer detailed trajectories of protein motions. Computational methods applied to X-ray crystallography and cryo-electron microscopy (cryo-EM) have enabled the exploration of protein dynamics, capturing conformational ensembles that were previously unattainable. The integration of machine learning, exemplified by AlphaFold2, has accelerated structure prediction and dynamics analysis. These approaches have revealed the importance of protein dynamics in allosteric regulation, enzyme catalysis, and intrinsically disordered proteins. The shift towards ensemble representations of protein structures and the application of single-molecule techniques have further enhanced our ability to capture the dynamic nature of proteins. Understanding protein dynamics is essential for elucidating biological mechanisms, designing drugs, and developing novel biocatalysts, marking a significant paradigm shift in structural biology and drug discovery. [ABSTRACT FROM AUTHOR]

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli , in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development. • AutoDC REdox robustly expresses VHHs. • 50% of VHHs expressed exceed the highest reported titer for cytoplasmic expression. • Consistent expression with minimal optimization across various VHH sequences. • Scalable fermentations up to a 1L bioreactor with VHH titers reaching up to 2 g/L. • Efficient autolysis and filtration process simplifies downstream purification. [ABSTRACT FROM AUTHOR]