The Protein Journal最新文献

Ataxin-2 (ATXN2), a key RNA-binding protein, regulates RNA metabolism, stress granule formation, and neuronal homeostasis, with dysregulated phosphorylation contributing to Spinocerebellar Ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and cancer. This review integrates structural biology, phosphoproteomics, and interactome analyses to map six critical phosphosites (S772, T741, S624, S684, S784, S889) within ATXN2’s intrinsically disordered regions. Modulated by kinases GSK3β and CDK13 and phosphatases like INPP5F, these sites orchestrate interactions with RNA-binding partners (e.g., ATXN2L, FXR2, STAU2) and co-regulated proteins (e.g., TP53BP1, NUP153), driving pathogenesis through disrupted autophagy, nucleocytoplasmic transport, and stress granule dynamics. We propose targeted therapies, including GSK3β inhibitors for ALS, antisense oligonucleotides for SCA2, and MTOR modulators for cancer, to restore ATXN2 function. By elucidating phosphocode of ATXN2, this work highlights novel avenues for precision medicine in neurodegenerative and oncogenic diseases.

6% hydroxyethyl starch (HES 130/0.4) is frequently employed to address hypovolemia, ensuring sufficient organ perfusion and oxygen transport. The effects on Apolipoprotein A-I (ApoA-I) were examined at three temperatures—280, 295, and 310 K—through several spectroscopic techniques to explore its possible interaction with the predominant protein in veins. The experimental findings indicated that HES 130/0.4 efficiently extinguished the intrinsic fluorescence of APOA-I. We also assessed the binding sites, binding constant, and thermodynamic parameters, which indicated that HES 130/0.4 can spontaneously associate with APOA-I via hydrogen bonds and van der Waals interactions (ΔG =  − 1.93 × 10⁴ J·mol⁻¹, ΔH =  − 5.63 × 10⁴ J mol⁻¹, and ΔS =  − 119 J mol⁻¹ K⁻¹) with a single binding site and week binding forces (n = 1.03 and K_A = 1.78 × 10³ M⁻¹) at body temperature. Moreover, the structure of APOA-I was significantly altered in the presence of HES 130/0.4. Blood Ca²⁺ and Fe³⁺ will diminish the storage duration. The study provides accurate and thorough foundational data to clarify the binding mechanisms of HES 130/0.4 with APOA-I in vitro, which may help the comprehension of its impact on protein function and toxic mechanism during transit and distribution in the bloodstream.

Nitrate contamination in water sources creates major health risks that primarily affect infants by causing methemoglobinemia (“blue baby syndrome”) while also leading to congenital defects and cancer development. The human body absorbs nitrates mainly through drinking contaminated water. Enzyme nitrate reductase (NR) produced by microorganisms, functions as a key factor in nitrate detoxification. A partial NR gene (GenBank accession: MN833805) from Aspergillus niger PKA16 (KY907172.1) was amplified by employing degenerate primers in this research. The primer sequences were designed based on conserved protein motifs and orthologous diversity analysis of 399 NR protein sequences spanning 127 fungal genera. The NR proteins exhibited an extensive range which demonstrated extensive intra- and interspecies diversity. The multiple conserved domains included nine motifs which remained consistent despite the observed sequence variability. Two highly conserved sequences RLTGKHPFN and PDHGYPLRLV were validated through degenerate-PCR which demonstrated their effectiveness for partial NR gene detection and amplification. In the present study, the developed degenerate primers enable researchers to detect and amplify NR genes from majority of known and unknown fungal strains including those identified through metagenomic studies also. This research establishes fundamental principles for using biotechnology to amplify bioremediatory enzyme nitrate reductase from fungal origin to clean up water and food that contains nitrates, to reduce the risk of ‘blue baby’ disease and cancer.

Bacterial antimicrobial resistance is a great public health threat worldwide, a situation that is much escalated by the rapid propagation of Extended Spectrum β-lactamase (ESBL) enzymes. These can hydrolyze and inactivate a broad range of β-lactams, including third-generation cephalosporins, penicillin, and aztreonam and are known to be associated with various bacterial infections, ranging from uncomplicated urinary tract infections to life-threatening sepsis.Variation is the essential raw material of Darwinian evolution and the accumulation of mutations plays one of the most important roles in it. Sequential acquisition of spontaneous mutations followed by successive rounds of selection can be attributed as one of the major reasons for the rapid diversification of ESBL enzymes. The ESBLs are excellent examples of ‘microevolution’ that led to ‘gain-of-function’ with an extended substrate spectrum. However, acquiring newer phenotypes sometimes comes with fitness costs and different mutational pathways interact with each other, triggering both additive and non-additive fitness to generate a rugged fitness landscape, that influences the path a strain must follow to adapt and evolve under selection pressure. Therefore, it is important to understand the role of mutations in the emergence of these enzyme variants. This review focuses on the understanding of different facades of mutational pathways that lead to the adaptive evolution of ESBL phenotype. The structural and mechanistic basis of the extension of the substrate spectrum by mutations are also discussed.

The bacterial HslVU enzyme complex consists of two components: the HslV protease and the HslU ATPase. This complex share both structural and sequence similarities with the eukaryotic proteasome. HslV becomes functionally active upon engagement with HslU, which inserts its C-terminal helix into a conserved groove within the HslV dimer. This interaction triggers allosteric modulation, thereby initiating HslV’s proteolytic activity. Because the HslVU system is present in pathogenic bacteria but absent in humans, it represents a promising target for antibacterial drug development. This study focuses on the discovery of small molecules that hyperactivate HslV, leading to excessive protein degradation in harmful bacterial strains. By integrating computational modeling with laboratory assays, four triazine-based compounds were identified as potent activators of HslV. These molecules demonstrated high binding affinity in docking simulations, favorable interaction profiles, and significant activation in biochemical assays. Their ED₅₀ values ranged from 0.37 μM to 0.55 μM, indicating strong potency. Furthermore, ADMET evaluations revealed desirable pharmacokinetic and drug-likeness properties. Overall, this work presents effective, non-peptidic small-molecule activators of the HslV protease and provides new insights into chemical modulation of the HslVU system, offering a promising avenue for antibacterial drug discovery.

Molecular chaperones known as heat shock proteins (HSPs) are evolutionarily conserved and are critical in regulating immune system functions. To assess the immunostimulatory potential of HSPs, we employed advanced computational and experimental techniques. Bioinformatics tools were employed to analyze and confirm the physicochemical characteristics of the full-length HSPA1A, the fragments of HSPA1A known as minichaperones (C-terminal region of HSPA1A: CHSPA1A and N-terminal region of HSPA1A: NHSPA1A), and the full length HSP27, confirming their advantageous properties and structural integrity through 3D refinement and validation. Docking studies revealed significant interactions of HSPs with CD91 and TLR4 receptors respectively, notably a strong and stable binding of the full length HSPA1A and CHSPA1A with CD91. Molecular dynamics simulations were conducted to confirm the structural stability of the complexes formed between HSPs and CD91. Immunological simulations demonstrated robust and stable responses from both innate and adaptive immune cells against HSPs. All HSPs were produced in E. coli, isolated via Ni-NTA affinity chromatography, and subsequently utilized to stimulate immune cells from mice in vitro. The results showed significant secretion of TNF-α and IFN-γ in comparison to IL-10, indicating a directed T-helper 1 (cellular) immunity. The in silico and in vitro findings represented that HSPA1A and then CHSPA1A resulted in significantly greater TNF-α secretion in splenocytes co-cultured with the pulsed DCs compared to NHSPA1A and HSP27, suggesting their significant role in activating adaptive immunity. In contrast, macrophages exhibited an increased TNF-α secretion in response to HSP27, suggesting its involvement in modulating innate inflammatory processes. Understanding these differences can help in designing more effective immunotherapies.

Homocysteine thiolactone is a reactive thiol known for its interaction with various proteins. Nevertheless, there exists a paucity of information concerning the interaction between homocysteine thiolactone and human insulin, particularly regarding the mechanism by which homocysteine facilitates the reduction of disulfide bonds within insulin. In the present study, we have elucidated the binding sites of homocysteine to the cysteine residues (A6-B7 and A20-B19) that are implicated in the formation of intermolecular disulfide bonds in insulin through an in vitro reaction analyzed via LC-ESI MS/MS. This results in a reduction of disulfide bonds linking the A and B chains, which was corroborated by MALDI-TOF-MS and ESI-MS analysis. The secondary structure of insulin is affected by this modification, as evidenced by circular dichroism spectroscopy. In-silico studies also show that homocysteine affects the insulin structure. A glucose uptake assay conducted in Chinese hamster ovary (CHO) cells that stably express the insulin receptor revealed that HC-modified insulin is less effective in inducing glucose uptake compared to native insulin, suggesting that HC-induced structural modifications in insulin influence functional activity. This study provides insight into the HC-induced structural and functional changes in insulin and discusses the consequent implications for diabetes.

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Superoxide dismutase (SOD) is found in a variety of organisms, including animals, plants, and microorganisms, and is widely used in medicine, food, and cosmetics. In this study, a novel heat-resistant SOD from Rhodothermus sp. XMH10 (RhSOD) has been found to have no loss of activity at 80 °C and exhibit high thermal stability across a temperature range from 20 °C to 80 °C. Unlike other reported SODs, RhSOD was found to have a unique small α-helix tail at the C-terminus, consisting of 11 amino acid residues. The absence of the C-terminal α-helix tail of RhSOD was shown to reduce its activity and thermal stability at 80 °C, suggesting that the C-terminal α-helix tail is crucial for the high thermal stability of RhSOD. Furthermore, the fusion of the C-terminal α-helix tail to the C-terminus of a thermophilic SOD from Anoxybacillus caldiproteolyticus (AcSOD) enhances its thermal stability at 70 °C and 80 °C. Circular dichroism (CD) spectral analysis further indicated that the C-terminal α-helix tail could improve the α-helix content, thus enhancing the structural stability of AcSOD. Thus, a novel C-terminal α-helix tail was firstly discovered, which could confer significant thermal stability to host proteins. This finding provides a new theoretical basis for the study of protein thermostability mechanism.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, encodes three small heat shock proteins (sHsps), PfHsp20a, PfHsp20b, and PfHsp20c, each containing a conserved α-crystallin domain (ACD). These class I sHsps are hypothesized to play critical roles in proteostasis under stress, yet their specific functions have remained poorly defined. In this study, all three sHsps were recombinantly expressed and purified for structural and functional characterization. Circular dichroism and thermal shift assays revealed distinct conformational properties, with PfHsp20a exhibiting the highest thermal and chemical stability. Functional assays using malate dehydrogenase and citrate synthase confirmed that all three isoforms possess autonomous chaperone activity, although with varying efficiency. Notably, the plant-derived flavonoid quercetin disrupted both the structure and function of the sHsps in a concentration-dependent manner, with PfHsp20c being the most sensitive. Quercetin also inhibited the growth of P. falciparum Nf54 and Dd2 strains in vitro with IC₅₀ values of 5.4 μM and 7.8 μM, respectively. These results provide the first direct evidence of independent chaperone activity in P. falciparum sHsps and highlight their vulnerability to small molecule inhibition. This establishes their potential as novel drug targets for antimalarial intervention.

The cohesin protein complex plays a very important role in chromosome segregation, transcription, DNA replication and chromosome condensation. Mutations in cohesin proteins give rise to a disease collectively referred to as Cohesinopathies. The major cause of cohesinopathies arise due to defects associated with gene expression, that give rise to developmental disorders. We have used Saccharomyces cerevisiae to mimic the cohesinopathy disorder Roberts syndrome with mutations (eco1W216G) homologous to that of humans (esco2). Our data suggests that polyol sugars like sorbitol, can repair misfolded proteins and reduce ER and proteostatic stress. We have used sorbitol as a chemical chaperone, to check how it can restore chromosome segregation, gene expression, misregulation, protein misfolding, autophagy and translational defects in the cohesin mutant of the Roberts’ phenotype. Molecular docking has helped us identify the possible sites on Eco1, which could possibly alter the phenotypic traits.