Protein Science最新文献

Histatin 5 (Hst5) is a 24-amino-acid peptide naturally present in human saliva that has been proposed as a potential antifungal therapeutic. However, Hst5 is susceptible to degradation by secreted aspartyl proteases (Saps) produced by Candida albicans, which could limit its efficacy as a therapeutic. To better understand the role of the lysine residues of Hst5 in proteolysis by C. albicans Saps (Sap1, Sap2, Sap3, Sap5, Sap6, Sap9, and Sap10), we studied variants of Hst5 with substitutions to leucine or arginine at the lysine residues (K5, K11, K13, and K17). Sap5, Sap6, and Sap10 did not degrade Hst5 or the variants. However, we observed degradation of the peptides by Sap1, Sap2, Sap3, and Sap9, and the degradation depended on the site of substitution and the substituent residue. Some modifications, such as K11L and K13L, were particularly susceptible to proteolysis by Sap1, Sap2, Sap3, and Sap9. In contrast, the K17L modification substantially increased the stability and antifungal activity of Hst5 in the presence of Saps. We used mass spectrometry to characterize the proteolysis products, which allowed us to identify fragments likely to have maintained or lost antifungal activity. We also evaluated the proteolytic stability of the Hst5 variants in saliva. Both K17L and K5R showed improved stability; however, the enhancements were modest, suggesting that further engineering is required to achieve significant improvements. Our approach demonstrates the potential of simple, rational substitutions to enhance peptide efficacy and proteolytic stability, providing a promising strategy for improving the properties of antifungal peptides.

Streptococcus pneumoniae (S. pneumoniae) employs various metabolic pathways to generate nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for redox balance, fatty acid synthesis, and energy production. GAPN, a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, plays a role in this process by directly reducing NADP⁺ to NADPH, effectively contributing to glucose metabolism. However, its relative importance for S. pneumoniae metabolism and infection has remained unknown. Here, we performed a comprehensive characterization of S. pneumoniae GAPN through kinetic assays, isothermal titration calorimetry (ITC), cryo-EM, mass spectrometry, and infection assays. Despite its structural similarities to its homologues in other species, S. pneumoniae GAPN exhibits negative cooperativity with respect to its substrate, glyceraldehyde-3-phosphate (G3P), suggesting a unique regulatory mechanism. Our results demonstrate that GAPN knockout leads to significant metabolic reprogramming, including increased glycogen storage that leads to enhanced fatty acid metabolism. This collectively reduces the ability of S. pneumoniae to manage oxidative stress and sustain infection. Our findings highlight GAPN as a critical enzyme for S. pneumoniae metabolic balance and suggest that its inhibition could serve as a potential strategy for therapeutic intervention in pneumococcal diseases.

G protein Coupled Receptors (GPCRs) are the largest family of cell surface receptors in humans. Somatic mutations in GPCRs are implicated in cancer progression and metastasis, but mechanisms are poorly understood. Emerging evidence implicates perturbation of intra-receptor activation pathway motifs whereby extracellular signals are transmitted intracellularly. Recently, sufficiently sensitive methodology was described to calculate structural strain as a function of missense mutations in AlphaFold-predicted model structures, which was extensively validated on experimental and predicted structural datasets. When paired with Molecular Dynamics (MD) simulations, these tools provide a facile approach to screen mutations in silico. We applied this framework to calculate the structural and dynamic effects of cancer-associated mutations in the chemokine receptor CCR3, a Class A GPCR involved in cancer and autoimmune disorders. Residue-residue contact scoring refined effective strain results, highlighting significant remodeling of inter- and intra-motif contacts along the highly conserved GPCR activation pathway network. We then integrated AlphaFold-derived predicted Local Distance Difference Test scores with per-residue Root Mean Square Fluctuations and activation pathway Contact Analysis (CONAN) from coarse grain MD simulations to identify statistically significant changes in receptor dynamics upon mutation. Finally, analysis of negative control mutants suggests false positive results in AlphaFold pipelines should be considered but can be mitigated with stricter control of statistical analysis. Our results indicate selected mutants influence structural plasticity of CCR3 related to ligand interaction, activation, and G protein coupling, using a framework that could be applicable to a wide range of biochemically relevant protein targets following further validation.

There is strong interest in accurate methods for predicting changes in protein stability resulting from amino acid mutations to the protein sequence. Recombinant proteins must often be stabilized to be used as therapeutics or reagents, and destabilizing mutations are implicated in a variety of diseases. Due to increased data availability and improved modeling techniques, recent studies have shown advancements in predicting changes in protein stability when a single-point mutation is made. Less focus has been directed toward predicting changes in protein stability when there are two or more mutations. Here, we analyze the largest available dataset of double point mutation stability and benchmark several widely used protein stability models on this and other datasets. We find that additive models of protein stability perform surprisingly well on this task, achieving similar performance to comparable non-additive predictors according to most metrics. Accordingly, we find that neither artificial intelligence-based nor physics-based protein stability models consistently capture epistatic interactions between single mutations. We observe one notable deviation from this trend, which is that epistasis-aware models provide marginally better predictions than additive models on stabilizing double point mutations. We develop an extension of the ThermoMPNN framework for double mutant modeling, as well as a novel data augmentation scheme, which mitigates some of the limitations in currently available datasets. Collectively, our findings indicate that current protein stability models fail to capture the nuanced epistatic interactions between concurrent mutations due to several factors, including training dataset limitations and insufficient model sensitivity.

Inherited mutations in the genes coding for the tumor suppressor proteins BRCA1 and PALB2 can lead to increased risk of breast and ovarian cancer. Upon DNA damage, these two proteins form a complex to promote double-stranded break repair via homologous recombination. Missense mutations in either BRCA1 or PALB2 that disrupt this important interaction result in loss of effective DNA damage repair and are associated with breast tumorigenesis. However, the overwhelming majority of missense mutations found in the binding domains of these two genes remain classified as variants of unknown significance. Here we report an in vitro assay for assessing the effect of variants of unknown significance on the heterodimerization of PALB2 and BRCA1 that recapitulates the effect of the known deleterious mutations. We apply the assay to several variants of unknown significance in BRCA1 which reveals other mutations in this region that also disrupt binding, including a mutation of a residue not predicted to directly interact with PALB2. Structural analysis indicates that all BRCA1 mutations to proline tested disrupt α-helix formation and therefore are not well tolerated even when located at positions outside of the PALB2-binding interface. This assay and the structural hypothesis described will be helpful for assessing risk for variants identified in the future in the BRCA1/PALB2 interaction domains.

Hsp90 is a dimeric molecular chaperone that is important for the folding, stabilization, activation, and maturation of hundreds of protein substrates called "clients" in cells. Dozens of co-chaperones and hundreds of post-translational modifications (PTMs) regulate the ATP-dependent client activation cycle. The Aha1 co-chaperone is the most potent stimulator of the ATPase cycle of Hsp90 and phosphorylation of threonine 22 in Hsp90 can regulate the recruitment of Aha1 in cells. We report here that phosphorylation of threonine 22 regulates specific aspects of Aha1 function after recruitment occurs. The phosphomimetic substitution, T22E, neutralizes the action of the Aha1 NxNNWHW motif. Moreover, this substitution can exert this effect from only one protomer of the Hsp90 dimer. This work sheds light on how asymmetric modifications in the Hsp90 dimer can functionalize individual protomers and fine-tune the Hsp90 cycle.

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions.

Cyclooxygenase-2 (COX-2) plays a crucial role in inflammation and has been implicated in cancer development. Understanding the behavior of COX-2 in different cellular contexts is essential for developing targeted therapeutic strategies. In this study, we investigate the fluorescence spectrum of a fluorogenic probe, NANQ-IMC6, when bound to the active site of human COX-2 in both its monomeric and homodimeric forms. We employ a multiscale first-principles simulation protocol that combines ground state MM-MD simulations with multiple excited state adiabatic QM/MM Born-Oppenheimer MD simulations based on linear response TD-DFT, which allows to account for protein heterogeneity effects on excited-state properties. Emission is then estimated from polarizable embedding TD-DFT QM/MMPol calculations. Our findings indicate that the emission shift arises from dimerization of the highly overexpressed COX-2 in cancer tissues, in contrast to the monomer structure present in inflammatory lesions and in normal cells with constitutive COX-2. This spectral shift is linked to changes in specific protein-probe interactions upon dimerization due to changes in the environment, whereas steric effects related to modulation of the NANQ geometry by the protein scaffold are found to be minor. This research paves the way for detailed investigations on the impact of environment structural transitions on the spectral properties of fluorogenic probes. Moreover, the fact that COX-2 exists as homodimer just in cancer tissues, but as monomer elsewhere, gives novel hints for therapeutical avenues to fight cancer and contributes to the development of drugs targeted to COX-2 dimer in cancer, but without affecting constitutive COX-2, thus minimizing off-target effects.

Robust and stable protein secretion is crucial for efficient recombinant protein production. Here, a novel and powerful platform using split GFP activated droplet sorting (SGADS) has been developed to significantly boost the yields of the protein of interest (POI). The SGADS platform leverages solubilizing peptide P17 and secretory expression in Bacillus subtilis to optimize two split GFP sensors: the P17-GFP1-9/GFP10-POI-GFP11 sensor for assessing protease activity and the P17-GFP1-10/GFP11-POI sensor for measuring secretion capacity. This innovative platform has demonstrated its effectiveness by successfully screening high-performance mutant strains capable of producing collagen, amylase, and protein glutaminase across a range of host organisms, including Escherichia coli, Bacillus subtilis, and Pichia pastoris. The substantial increases in production achieved with the SGADS platform highlight its broad applicability and potential in enhancing recombinant protein production.