Search Results (59)

Methacrylyl-CoA is a key metabolic intermediate in the valine catabolic pathway. Its accumulation has been found to be cytotoxic and associated with pathological conditions. Nevertheless, detailed biological effects of methacrylyl-CoA and methacrylate in human physiology and pathology are poorly understood. We propose that the electrophilicity of the alkene bond in the methacrylyl group can react with the cysteine residues in proteins resulting in an unexplored protein post-translational modification (PTM), cysteine S-2-carboxypropylation (C2cp). To test and validate this mechanistic hypothesis, we experimentally detected and profiled S-2-carboxypropylated proteins from the complex cellular proteome with the design and application of a bioorthogonal chemical probe, N-propargyl methacrylamide. We tested the probe in different mammalian cell models and demonstrated its versatility and sensitivity to protein cysteine S-2-carboxypropylation. We established quantitative chemical proteomics for global and site-specific profiling of protein S-2-carboxypropylation, which successfully identified 403 S-2-carboxypropylated proteins and 120 cysteine modification sites from HEK293T cells. Through bioinformatic analysis, we found that C2cp-modified proteins were involved in a variety of critical cellular functions including translation, RNA splicing, and protein folding. Our chemoproteomic studies demonstrating the proteome-wide distribution of cysteine S-2-carboxypropylation provide a new biochemical mechanism for the functional investigation of methacrylyl-CoA and understanding valine-related metabolic disorders. Full article

Rhodopsin, the prototypical Class A G protein-coupled receptor (GPCR) and visual pigment of rod photoreceptors, has long served as a structural and mechanistic model for GPCR biology. Mutations in rhodopsin are the leading cause of autosomal dominant retinitis pigmentosa (adRP), making this receptor a critical therapeutic target. In this review, we summarize the chemical, structural, and biophysical features of small-molecule modulators of this receptor, spanning both classical retinoid analogs and emerging non-retinoid scaffolds. These ligands reveal recurrent binding modes within the orthosteric chromophore pocket as well as peripheral allosteric and bitopic sites, where they mediate folding, rescue trafficking, photocycle modulation, and mutant stabilization. We organize ligand performance into a three-tier framework linking binding affinity, cellular rescue potency, and stability gains. Chemotypes in tier 2, which show sub-micromolar to low-micromolar activity with broad mutant coverage, emerge as promising candidates for optimization into next-generation scaffolds. Across scaffolds, a recurring minimal pharmacophore is evident by a contiguous hydrophobic π-surface anchored in the β-ionone region, coupled with a strategically oriented polar handle that modulates the Lys296/Glu113 microenvironment, offering tractable design vectors for non-retinoid chemotypes. Beyond the chromophore binding pocket, we highlight opportunities to exploit extracellular loop epitopes, cytoplasmic microswitch clefts, dimer/membrane interfaces, and ion co-binding sites to engineer safer, state-biased control with fewer photochemical liabilities. By integrating rhodopsin photobiophysics with environment-aware, multi-state medicinal chemistry, and by addressing current translational challenges in drug delivery, this review outlines a rational framework for advancing rhodopsin-targeted therapeutics toward clinically credible interventions for RP and related retinal degenerations. Full article

The liver orchestrates metabolic homeostasis through dynamic post-translational modifications. β-hydroxybutyrylation (Kbhb), a ketone body-driven modification, regulates epigenetics and metabolism in humans and mice but remains unexplored in livestock. Here, we characterize the porcine hepatic β-hydroxybutyrylome using high-resolution mass spectrometry, identifying 4982 Kbhb sites on 2122 proteins—the largest dataset to date. β-hydroxybutyrylation predominantly targets non-histone proteins (99.68%), with enrichment in fatty acid β-oxidation, TCA cycle, and oxidative phosphorylation pathways. Subcellular localization revealed cytoplasmic (38.1%), mitochondrial (18.1%), and nuclear (15.3%) dominance, reflecting BHB-CoA synthesis sites. Motif analysis identified conserved leucine, phenylalanine, and valine residues at modified lysines, suggesting enzyme-substrate specificity. β-hydroxybutyrate treatment elevated global Kbhb levels, increasing TCA intermediates (e.g., α-ketoglutarate, +9.56-fold) while reducing acetyl-CoA, indicating enhanced mitochondrial flux. Cross-species comparisons showed tissue-specific Kbhb distribution (nuclear in human cells vs. mitochondrial in mice), highlighting metabolic adaptations. This study establishes pigs as a model for Kbhb research, linking it to energy regulation and providing insights into metabolic reprogramming. Full article

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions. Full article

Background: A considerable proportion of the symptoms associated with excessive dietary intake can be attributed to systemic imbalances in lipid metabolism. The prominent toxicity of saturated fatty acids has been repeatedly demonstrated and sheds light on the protective role of stearoyl-CoA desaturase-1 (SCD1), the key enzyme for fatty acid desaturation. SCD1 protein expression is regulated at the levels of transcription, translation, and degradation. However, the modulating effect of the variability of the human genome must also be taken into account. Therefore, we aimed to ascertain whether natural missense or frameshift mutations in SCD1 (p.H125P, p.M224L, p.A333T, p.R253AfsTer7) could influence the expression, degradation, or function of the enzyme. Methods: In silico and in vitro experiments were conducted to comprehensively evaluate the consequences associated with each genetic variation, with the objective of using the results to propose a risk or severity ranking of SCD1 variants. Results: As anticipated, the p.R253AfsTer7 variant was identified as the most deleterious in structural, functional, and quantitative terms. The p.H125P variant also reduced the desaturation capacity of the enzyme in accordance with the predicted structural alterations and augmented degradation resulting from folding complications. This was aggravated by increased mRNA instability and accompanied by mild endoplasmic reticulum stress induction. The p.A333T protein exhibited an intermediate phenotype, whereas p.M224L showed no deleterious effects and even increased the amount of SCD1. Conclusions: In conclusion, the large-scale identification of genetic variations needs to be supplemented with comprehensive functional characterization of these variations to facilitate adequate personalized prevention and treatment of lipid metabolism-related conditions. Full article

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)₂ was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR’s strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines. Full article

By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents. Full article

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson’s disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases. Full article

Extensive evidence supports the connection between obesity-induced inflammation and the heightened expression of IL-6 adipose tissues. However, the mechanism underlying the IL-6 exacerbation in the adipose tissue remains unclear. There is general agreement that TNF-α and stearate concentrations are mildly elevated in adipose tissue in the state of obesity. We hypothesize that TNF-α and stearate co-treatment induce the increased expression of IL-6 in mouse adipocytes. We therefore aimed to determine IL-6 gene expression and protein production by TNF-α/stearate treated adipocytes and investigated the mechanism involved. To test our hypothesis, 3T3-L1 mouse preadipocytes were treated with TNF-α, stearate, or TNF-α/stearate. IL-6 gene expression was assessed by quantitative real-time qPCR. IL-6 protein production secreted in the cell culture media was determined by ELISA. Acetylation of histone was analyzed by Western blotting. Il6 region-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac) was determined by ChIP-qPCR. 3T3-L1 mouse preadipocytes were co-challenged with TNF-α and stearate for 24 h, which led to significantly increased IL-6 gene expression (81 ± 2.1 Fold) compared to controls stimulated with either TNF-α (38 ± 0.5 Fold; p = 0.002) or stearate (56 ± 2.0 Fold; p = 0.013). As expected, co-treatment of adipocytes with TNF-α and stearate significantly increased protein production (338 ± 11 pg/mL) compared to controls stimulated with either TNF-α (28 ± 0.60 pg/mL; p = 0.001) or stearate (53 ± 0.20 pg/mL, p = 0.0015). Inhibition of histone acetyltransferases (HATs) with anacardic acid or curcumin significantly reduced the IL-6 gene expression and protein production by adipocytes. Conversely, TSA-induced acetylation substituted the stimulatory effect of TNF-α or stearate in their synergistic interaction for driving IL-6 gene expression and protein production. Mechanistically, TNF-α/stearate co-stimulation increased the promoter-associated histone H3 lysine 9/18 acetylation (H3K9/18Ac), rendering a transcriptionally permissive state that favored IL-6 expression at the transcriptional and translational levels. Our data represent a TNF-α/stearate cooperativity model driving IL-6 expression in 3T3-L1 cells via the H3K9/18Ac-dependent mechanism, with implications for adipose IL-6 exacerbations in obesity. Full article

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described. Full article

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine. Full article

The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNa_V1.4 with a 939 nM IC₅₀. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity. Full article

Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A’s N terminal LRR domain. Full article

Molecular dynamics simulations of protein folding typically consider the polypeptide chain at equilibrium and in isolation from the cellular components. We argue that in order to understand protein folding as it occurs in vivo, it should be modeled as an active, energy-dependent process, in which the cellular protein-folding machine directly manipulates the polypeptide. We conducted all-atom molecular dynamics simulations of four protein domains, whose folding from the extended state was augmented by the application of rotational force to the C-terminal amino acid, while the movement of the N-terminal amino acid was restrained. We have shown earlier that such a simple manipulation of peptide backbone facilitated the formation of native structures in diverse α-helical peptides. In this study, the simulation protocol was modified, to apply the backbone rotation and movement restriction only for a short time at the start of simulation. This transient application of a mechanical force to the peptide is sufficient to accelerate, by at least an order of magnitude, the folding of four protein domains from different structural classes to their native or native-like conformations. Our in silico experiments show that a compact stable fold may be attained more readily when the motions of the polypeptide are biased by external forces and constraints. Full article

Entangled motifs are found in one-third of protein domain structures, a reference set that contains mostly globular proteins. Their properties suggest a connection with co-translational folding. Here, we wish to investigate the presence and properties of entangled motifs in membrane protein structures. From existing databases, we build a non-redundant data set of membrane protein domains, annotated with the monotopic/transmembrane and peripheral/integral labels. We evaluate the presence of entangled motifs using the Gaussian entanglement indicator. We find that entangled motifs appear in one-fifth of transmembrane and one-fourth of monotopic proteins. Surprisingly, the main features of the distribution of the values of the entanglement indicator are similar to the reference case of general proteins. The distribution is conserved across different organisms. Differences with respect to the reference set emerge when considering the chirality of entangled motifs. Although the same chirality bias is found for single-winding motifs in both membrane and reference proteins, the bias is reversed, strikingly, for double-winding motifs only in the reference set. We speculate that these observations can be rationalized in terms of the constraints exerted on the nascent chain by the co-translational bio-genesis machinery, which is different for membrane and globular proteins. Full article