Protein Science最新文献

In preclinical models, Phosphodiesterase-10A (PDE10A) inhibition has shown efficacy for Parkinson's and Huntington's diseases and is potentially a therapeutic approach for schizophrenia. In this study, computational approaches were employed to identify selective PDE10A inhibitors from a series of olefinated benzosuberene analogues. The molecular interactions and docking scores of the selected molecules were compared with three different co-crystal inhibitors of PDE10A. Molecular dynamics (MD) simulations confirmed the stability of the screened hit molecules with PDE10A, and AMKPD-52 was found to uniquely interact with the selectivity pocket residue Tyr-683. Further refinement using steered MD simulations and umbrella sampling simulations with a total simulation time of ~72 μs reinforced AMKPD-52 as the potential lead molecule. Absolute binding free energy calculations performed using multistate Bennett acceptance ratio method consistently ranked AMKPD-52 as the potential lead molecule for PDE10A inhibition. Experimental evaluation revealed an IC₅₀ value of 11.52 μM for AMKPD-52, confirming its inhibitory activity against PDE10A. Although the binding affinity and potency were modest compared to the reference inhibitor TAK-063, the natural origin and straightforward synthetic tractability of AMKPD-52 make it an attractive candidate for further medicinal chemistry optimization. These findings warrant in vivo validation to assess the therapeutic potential of AMKPD-52 in PDE10A-targeted interventions.

Until today, the exact function of mammalian odorant binding proteins (OBPs) remains a topic of debate. Although their main established function lacks direct evidence in human olfaction, OBPs are traditionally believed to act as odorant transporters in the olfactory sense. Now, available RNA-seq and proteomics data identified the expression of the two known human OBPs (hOBP2A and hOBP2B) in both male and female reproductive tissues. Therefore, we hypothesize that OBPs may possess functions that go beyond the olfactory sense, potentially as hormone transporters. Such a function could further link them to the tumorigenesis and cancer progression of hormone-dependent cancer types including ovarian, breast, prostate, and uterine cancer. In this structured review, we use available data to explore the effects of genetic alterations such as somatic copy number aberrations and single nucleotide variants on OBP function and their corresponding gene expression profiles. Our computational analyses suggest that somatic copy number aberrations in OBPs are associated with large changes in gene expression in reproductive cancers while point mutations have little to no effect. Additionally, the structural characteristics of OBPs, together with other lipocalin family members, allow us to explore putative functions within the context of cancer biology. Our overview consolidates current knowledge on putative human OBP functions, their expression patterns, and structural features. Finally, it provides an overview of applications, highlighting emerging hypotheses and future research directions within olfactory and non-olfactory roles.

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised the efficacy of many authorized monoclonal antibody products. This highlights the need for alternative strategies, especially for vulnerable populations such as immunocompromised individuals. Here, we optimized angiotensin-converting enzyme 2 (ACE2)-Fc fusion proteins by combining three engineering steps: in silico mutagenesis of the S protein binding interface to increase affinity, insertion of a flexible linker to improve protein stability and S protein accessibility, and generation of a tetrameric molecule to maximize avidity. Neutralizing activity was tested against a large panel of pre-Omicron and Omicron pseudoviruses and authentic viruses, including JN.1 and KP.2 variants. Optimized ACE2-Fc molecules demonstrated potent neutralizing activity, in the picomolar range, against all SARS-CoV-2 variants. Our molecules displayed similar potency but better resilience when compared to the monoclonal antibody Sipavibart. These findings support ACE2-Fc proteins as robust candidates for next-generation interventions against infection by an evolving SARS-CoV-2.

Avian eggshell membrane (ESM) is fabricated within the isthmus region of the oviduct and is comprised of three juxtaposed, predominantly proteinaceous layers lying between egg white and the calcified shell. The limiting membrane is less than 0.5 μm in thickness and forms the osmotic barrier for the egg. This first layer provides the foundation for the successive deposition of two mats of protein fibers. Fibers from both inner and outer layers appear to have similar amino acid compositions and are notably disulfide-rich (comprising about 10% Cys). ESM has been utilized in a wide variety of applications, including nutraceutical supplements, tissue engineering, and nanofabrication, and yet fundamental questions concerning protein composition, fiber structure, and membrane assembly remain to be resolved. We previously identified an abundant disulfide-rich structural protein in chicken ESM fibers (cysteine-rich eggshell membrane protein; CREMP) that contains multiple tandemly repeated modules. In this work, we determine a structural model for four consecutive 2-disulfide containing CREMP modules using a variety of two- and three-dimensional solution NMR experiments. CREMP modules feature an N-terminal loop region positioned above a small beta hairpin that is stabilized by a conserved pattern of disulfide bridges between Cys1-3 and Cys2-4. While the individual CREMP modules are highly ordered, the lack of long-range inter-module restraints suggests an extended structure connected by flexible linkers. Finally, the structural information obtained in this work is considered in the context of full-length CREMP proteins and compared to two other structural proteins that contain multiple tandem repeats of 2-disulfide modules.

Macromolecular crowding is ubiquitous in physiological environments, perturbing the thermodynamics and kinetics of proteins via excluded volume and nonspecific chemical interactions. While crowding has been well studied in vitro and in cells, the inert sugar polymers used to simulate crowding lack the chemical characteristics of biomolecules. Emerging studies guide the development of more relevant models of crowding in the cell, but little work has been done to discern crowding effects on proteins at the cell surface. Using ¹⁹F NMR, we measure how protein stability, folding, and intermolecular interactions are modulated by three glycopolymers abundant at the cellular exterior. Biologically relevant glycopolymers including heparin, hyaluronic acid, and mucin significantly stabilize the folding of the N-terminal domain of the Drk-SH3 protein. These interactions are enthalpically stabilizing, emphasizing the importance of chemical interactions for biologically relevant crowders. We further show that these glycopolymers stabilize a homodimer formed by the A34F variant of GB1, demonstrating that biological crowders not only affect isolated proteins, but also influence how proteins interact with one another. Crowding is more complex than simple ideas of volume exclusion suggest, and our work guides a more comprehensive understanding of protein crowding in the context of the glycocalyx, the last frontier of the cell.

High-temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram-negative and human HtrAs are structurally well studied, Gram-positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram-positive HtrA, from Streptococcus pneumoniae, a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram-positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo-EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA-PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram-positive HtrAs as a mechanistically divergent subgroup within the HtrA family.

Liquid-liquid phase separation (LLPS) is emerging as a key mechanism for organizing cellular components and regulating stress responses. Although LLPS has been extensively studied in intrinsically disordered proteins, whether highly charged and intrinsically disordered molecular chaperones undergo LLPS remains poorly understood. Here, we demonstrate that the Escherichia coli acid shock protein Asr, a highly charged and intrinsically disordered chaperone, undergoes LLPS driven by electrostatic interactions and forms dynamic liquid condensates with polyanions such as DNA, RNA, heparin, and acidic proteins. Asr phase separation critically depends on positively charged clusters, polyanion length, ionic strength, and pH. Guided by Asr's physicochemical features, we identify three additional molecular chaperones, Anhydrin, Hero7, and HCVncd, that also exhibit LLPS behavior in vitro but display distinct condensate properties and pH responsiveness consistent with their individual charge compositions and distributions. In vivo, Asr-EGFP forms non-canonical compartments in 37% of E. coli cells at pH 7.5, increasing to 80% under acidic conditions (pH 4.5). These compartments disassemble under high-salt conditions after cell lysis, suggesting electrostatic mediation. In cell imaging and FRAP analyses further reveal that charge-enhanced Asr mutants and homologs form canonical condensates in vivo, predominantly co-localizing with acidic proteins. Notably, Asr*3 fusion drives condensate formation of the aggregation-prone client thereby reducing stress-induced aggregation, indicating that Asr functions as an LLPS-promoting module to mitigate protein aggregation. These findings advance our understanding of LLPS in highly charged, intrinsically disordered molecular chaperones and lay the foundation for exploring their roles in cellular homeostasis and potential applications in engineering synthetic biomolecular condensates.

Curli, which are the major proteinaceous components of the Escherichia coli biofilm extracellular matrix, help protect cells against environmental stressors, including dehydration and antibiotics. Composed of the amyloid proteins CsgA and CsgB, curli self-assemble as these protomers are secreted into the extracellular space. The mechanisms of curli assembly and their functional roles within the extracellular matrix are incompletely understood. High-resolution imaging tools compatible with live-cell conditions provide a critical means to investigate the assembly and function of curli in their native context. In this study, we use super-resolution imaging to visualize curli fibrils on living bacterial cells. Transient amyloid binding of the fluorogenic dye Nile blue facilitates two complementary super-resolution fluorescence microscopy approaches: single-molecule imaging via points accumulation for imaging in nanoscale topography and super-resolution optical fluctuation imaging via pixel-wise autocorrelation. Additionally, imaging fluorescence correlation spectroscopy was used to measure the characteristic relaxation times associated with Nile blue binding to CsgA fibrils. Together, these approaches offer a framework for imaging-based biophysical characterization of curli structures on living cells.

OX40 and OX40L belong to the tumor necrosis factor receptor superfamily (TNFRSF) and tumor necrosis factor superfamily (TNFSF), respectively. Protein-protein interactions between OX40 and OX40L facilitate T cell responses, triggering various immunological and pathophysiological events. Excessive activation frequently contributes to the onset of autoimmune and allergic diseases. Therefore, the OX40/OX40L system is considered a promising target for drug discovery. Given that the structure of the OX40-OX40L complex exhibits some unique features compared to other members of these protein super families, it is reasonable to assume that this tandem possesses distinct interaction mechanisms. However, detailed interaction analysis using quantitative parameters such as binding kinetics or thermodynamics, with remains to be performed for OX40/OX40L. In this study, we identified several hot spot residues from the OX40 cysteine-rich domains (CRDs) 1 to 3 by alanine scanning. Kinetic and thermodynamic analysis combined with molecular dynamics simulations highlighted the characteristics of a hot spot from CRD3 due to its indirect influence on those from CRD1 and CRD2, providing insights into the interaction mechanism and a strategy for drug discovery targeting this interaction.

Eukaryotic elongation factor 2 kinase (eEF-2K) is a member of the α-kinase family of atypical serine/threonine kinases. eEF-2K, the only calmodulin-activated α-kinase, phosphorylates the ribosome-associated GTPase, eukaryotic elongation factor 2 (eEF-2), suppressing translational elongation. α-kinases, including eEF-2K, possess catalytic site geometries that are distinct from those of typical kinases, suggesting possible divergence in their phospho-transfer mechanisms. Unlike typical protein kinases, where chemistry is known to proceed through a sequential mechanism involving a ternary kinase-substrate-ATP•Mg²⁺ complex, the nature of the chemical step catalyzed by α-kinases remains poorly defined. Here, multiple orthogonal lines of evidence, including a crystal structure and solution-state mass spectrometry data, suggest phosphorylation of a catalytically essential aspartate residue (D284) at the eEF-2K active site. Previous crystallographic evidence of the presence of a phospho-aspartate at an equivalent position (D766) in the related Dictyostelium α-kinase MHCK-A strongly suggests that this species represents a conserved active-site feature in α-kinase family members, despite their disparate modes of activation. This observation, together with existing kinetics data on eEF-2K, raises the possibility that phospho-transfer chemistry in α-kinases occurs via an ordered stepwise mechanism involving a phospho-enzyme intermediate, contrasting with typical protein kinases.