Search Results (58)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread enzymopathy affecting approximately 500 million individuals that represents a significant global health issue. Among the more than 230 identified mutations in the G6PD gene, six class A variants—G6PD Utrecht (Pro409Ser), G6PD Suwalki (Pro409Arg), G6PD Merlo (Pro409Gln), G6PD Kawasaki (Gly410Ala), G6PD Shinagawa (Gly410Asp), and G6PD Riverside (Gly410Cys)—are located in the beta-loop near the NADP⁺ binding site. These mutations are of particular interest due to their association with severe hematologic phenotypes, including chronic hemolytic anemia, as well as their proposed role in the allosteric regulation of G6PD multimerization. This study presents a comprehensive biochemical and functional characterization of these clinically relevant G6PD variants. The variant enzymes were cloned, expressed, and purified for characterization. Kinetic parameters and thermal stability assays, complemented by molecular dynamics simulations (MDS), were employed to elucidate the structural impacts of the mutations. Our results demonstrate that these mutations significantly impair protein function, characterized by reduced affinity for glucose-6-phosphate (G6P) and NADP⁺, as well as altered thermal stability compared with wild-type G6PD. MDS revealed that point mutations in the βN- and βM-sheets in the NADP⁺_s region propagate subtle conformational changes, ultimately affecting the NADP⁺c region and the G6P binding cavity. Furthermore, secondary structure element analyses of the simulation data showed that Pro409 and Gly410 point mutations propagate several changes around residues 195–210 (G6P binding site) and 380–400 (NADP⁺_s), explaining their effect on overall catalytic performance. These findings enhance our understanding of the molecular mechanisms underlying G6PD deficiency and its clinical implications, providing a foundation for future therapeutic strategies aimed at mitigating the effects of these variants. Full article

Enhancing protein–protein interactions is a key therapeutic strategy to ensure effective protein function in terms of pharmacokinetics and pharmacodynamics and can be accomplished with methods like directed evolution or rationale design. Previously, two papers suggested the possible enhancement of protein–protein binding affinity via destabilizing mutations. This paper reviews the results of the previous literature and adds new data to show the generality of the strategy that destabilizing the unbound protein without significantly changing the free energy of the complex can enhance protein–protein interactions for therapeutic benefit. The first example presented is that of a variant of human growth hormone (hGHv) containing 15 mutations that improve the binding to the hGH binding protein (hGHbp) by 400-fold while retaining full biological activity. The second example is that of the YTE mutations (M252Y/S354T/T256E) in the Fc region of a monoclonal antibody (mAb). The YTE mutations improve the binding of the mAb to FcRn at pH 6.0 10-fold, resulting in elongated serum half-life of the mAb. In both cases, (i) chemical titration or differential scanning calorimetry (DSC) showed the mutations destabilize the unbound mutant proteins, (ii) isothermal titration calorimetry (ITC) showed extremely favorable enthalpy (ΔH) and unfavorable entropy (ΔS) upon binding to their respective target molecule compared with the wildtype, and (iii) hydrogen/deuterium exchange–mass spectrometry (HDX-MS) revealed that these mutations increase the free energy of unbound mutant protein without significantly affecting the free energy of the bound state, resulting in an enhancement to the binding affinities. The third example presented is that of the JAWA mutations (T437R/K248E) also located in the Fc region of a mAb. The JAWA mutations facilitate antibody multimerization upon binding to cell surface antigens, allowing for enhanced agonism and effector functions. Both DSC and HDX-MS showed that the JAWA mutations destabilize the unbound Fc, although the complex was not characterized due to weak binding. Enhancement of protein–protein interactions through incorporation of mutations that increase the free energy of a protein’s unbound state represents an alternative route to decreasing the protein–protein complex free energy through optimization of the binding interface. Full article

Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article

SYPRO^® Orange (SyO) is a zwitterionic dye that is used for protein gel staining, for thermal melt assays of proteins, and as a marker for misfolded proteins. However, while widely utilized, much of SyOs’ photophysics remains unexplored. We studied the effect of pH on the photophysical properties of SyO in aqueous solution and found two well-defined transitions in the 0 to 10 pH range between three SyO species with distinct absorption and fluorescence properties. The first transition occurs around pH 1.5 and appears to be a coupled deprotonation–aggregation event. The second transition occurs between pH 4 and 5, and its pK_a depends on the concentration of SyO. A link between the concentration dependence of the pK_a of the second pH transition and the aggregation behavior of SyO at neutral pH is discussed, and aggregation equilibrium titrations are presented that suggest that SyO forms multimeric aggregates at neutral pH containing ten or more SyO molecules. Full article

Depositions of protein aggregates are typical pathological hallmarks of various neurodegenerative diseases (NDs). For example, amyloid-beta (Aβ) and tau aggregates are present in the brain and plasma of patients with Alzheimer’s disease (AD); α-synuclein in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA); mutant huntingtin protein (Htt) in Huntington’s disease (HD); and DNA-binding protein 43 kD (TDP-43) in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). The same misfolded proteins can be present in multiple diseases in the form of mixed proteinopathies. Since there is no cure for all these diseases, understanding the mechanisms of protein aggregation becomes imperative in modern medicine, especially for developing diagnostics and therapeutics. A Multimer Detection System (MDS) was designed to distinguish and quantify the multimeric/oligomeric forms from the monomeric form of aggregated proteins. As the unique epitope of the monomer is already occupied by capturing or detecting antibodies, the aggregated proteins with multiple epitopes would be accessible to both capturing and detecting antibodies simultaneously, and signals will be generated from the oligomers rather than the monomers. Hence, MDS could present a simple solution for measuring various conformations of aggregated proteins with high sensitivity and specificity, which may help to explore diagnostic and treatment strategies for developing anti-aggregation therapeutics. Full article

Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization. We observed that the psi (Ψ) packaging signal and the dimerization initiation sequence (DIS) had stabilizing effects on RSV Gag condensates, while RNAs lacking these features promoted or antagonized condensation, depending on local protein concentration and condensate architecture. An RNA containing Ψ, DIS, and the dimerization linkage structure (DLS) that is capable of stable dimer formation was observed to act as a bridge between RSV Gag condensates. These observations suggest additional, condensate-related roles for Gag-Ψ binding, gRNA dimerization, and Gag dimerization/multimerization in gRNA selection and packaging, representing a significant step forward in our understanding of how these interactions collectively facilitate efficient genome packaging. Full article

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamD^Y184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamD^R94, BamD^R97, and BamA^E127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex. Full article

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H⁺ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction. Full article

Myocardial ischaemia reperfusion injury (IRI) occurring from acute coronary artery disease or cardiac surgical interventions such as bypass surgery can result in myocardial dysfunction, presenting as, myocardial “stunning”, arrhythmias, infarction, and adverse cardiac remodelling, and may lead to both a systemic and a localised inflammatory response. This localised cardiac inflammatory response is regulated through the nucleotide-binding oligomerisation domain (NACHT), leucine-rich repeat (LRR)-containing protein family pyrin domain (PYD)-3 (NLRP3) inflammasome, a multimeric structure whose components are present within both cardiomyocytes and in cardiac fibroblasts. The NLRP3 inflammasome is activated via numerous danger signals produced by IRI and is central to the resultant innate immune response. Inhibition of this inherent inflammatory response has been shown to protect the myocardium and stop the occurrence of the systemic inflammatory response syndrome following the re-establishment of cardiac circulation. Therapies to prevent NLRP3 inflammasome formation in the clinic are currently lacking, and therefore, new pharmacotherapies are required. This review will highlight the role of the NLRP3 inflammasome within the myocardium during IRI and will examine the therapeutic value of inflammasome inhibition with particular attention to carbon monoxide, nitric oxide, and hydrogen sulphide as potential pharmacological inhibitors of NLRP3 inflammasome activation. Full article

Reliable and accurate methods of estimating the accuracy of predicted protein models are vital to understanding their respective utility. Discerning how the quaternary structure conforms can significantly improve our collective understanding of cell biology, systems biology, disease formation, and disease treatment. Accurately determining the quality of multimeric protein models is still computationally challenging, as the space of possible conformations is significantly larger when proteins form in complex with one another. Here, we present EGG (energy and graph-based architectures) to assess the accuracy of predicted multimeric protein models. We implemented message-passing and transformer layers to infer the overall fold and interface accuracy scores of predicted multimeric protein models. When evaluated with CASP15 targets, our methods achieved promising results against single model predictors: fourth and third place for determining the highest-quality model when estimating overall fold accuracy and overall interface accuracy, respectively, and first place for determining the top three highest quality models when estimating both overall fold accuracy and overall interface accuracy. Full article

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein–protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy. Full article

Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have more than one active state, a phenomenon called either ‘biased agonism’, ‘functional selectivity’, or ‘ligand-directed signaling’. However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called ‘biased modulation’, that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between neuraminidase-1 (Neu-1) and matrix metalloproteinase-9 (MMP-9) in the activation of glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Aravnil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue, PANC-1, and SW-620 cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link that regulates the interaction and signaling mechanism among these molecules present on the cell surface. Moreover, the study results demonstrate that CB1 agonists induce NFκB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial–mesenchymal markers, E-cadherin, and vimentin in SW-620 cells, albeit the impact on E-cadherin expression is less pronounced compared to vimentin. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis. Full article

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies. Full article

COCH (coagulation factor C homology) is one of the most frequently mutated genes of autosomal dominant non-syndromic hearing loss. Variants in COCH could cause DFNA9, which is characterized by late-onset hearing loss with variable degrees of vestibular dysfunction. In this study, we report a Chinese family with a novel COCH variant (c.1687delA) causing p.D544Vfs*3 in the cochlin. Comprehensive audiometric tests and vestibular function assessments were taken to acquire the phenotypic profile of the subjects. Next-generation sequencing was conducted and segregation analysis was carried out using Sanger sequencing. The proband presented mild vestibular symptoms and normal functional assessment results in almost every test, while the variant co-segregated with hearing impairment in the pedigree. The variant was located beyond the vWFA2 domain, which was predicted to affect the post-translational cleavage of the cochlin via molecular modeling analysis. Notably, in the overexpressing study, by transient transfecting the HEK 293T cells, we found that the p.D544Vfs*3 variant increased the formation of multimeric cochlin. Our result enriched the spectrum of DFNA9-linked pathological COCH variants and suggested that variants, causative of cochlin multimerization, could be related to DFNA9 with sensorineural hearing loss rather than serious vestibular symptoms. Full article

Semaphorins belong to a group of membrane and secretory proteins that act as ligands for several receptor families and are involved in modulating cell signaling pathways. They bind multimeric receptor complexes on the cell membrane to exert their effects and initiate unique intracellular signal transduction cascades. These proteins can influence several processes that are very important for cell function, such as cell division and differentiation. Semaphorins are involved in cell migration, apoptosis, cell adhesion, aggregation, and numerous immune processes due to their immunoregulatory effects. Semaphorins are expressed in keratinocytes, which is why they have become a target for studies on the pathogenesis of skin diseases. Most studies to date on the role of semaphorins in the pathogenesis of skin diseases have been carried out in cellular or animal models, and there are few clinical studies evaluating the role of semaphorins in the pathogenesis and therapy of skin diseases. In this narrative review, we summarized the current state of knowledge on the role of semaphorins in the pathogenesis of skin diseases and their potential importance as targets for therapy. We also tried to present the key findings and weaknesses of previous research in this field. The novelty of this article lies in the comprehensive presentation of the role of semaphorins in the pathogenesis of skin diseases, including the results of studies on cell cultures and animal models, elucidating the mechanisms and signaling pathways through which semaphorins affect the development of skin diseases, as well as on the presentation of the results of existing clinical trials evaluating the role of semaphorins in the pathogenesis of skin diseases, and as potential therapeutic targets. Full article