Adrenal pheochromocytoma impacts three main pathways: cysteine-methionine, pyrimidine, and tyrosine metabolism. / è‚¾ä¸Šè ºå—œé“¬ç»†èƒžç˜¤å½±å“ä¸‰æ¡ä¸»è¦ä»£è°¢é€šè·¯ï¼šåŠèƒ±æ°¨é ¸-è›‹æ°¨é ¸ä»£è°¢ã€å˜§å•¶ä»£è°¢å’Œé ªæ°¨é ¸ä»£è°¢é€šè·¯.

Pheochromocytomas and paragangliomas (PPGLs) cause symptoms by altering the circulation levels of catecholamines and peptide hormones. Currently, the diagnosis of PPGLs relies on diagnostic imaging and the detection of catecholamines. In this study, we used ultra-performance liquid chromatography (UPLC)/quadrupole time-of-flight mass spectrometry (Q-TOF MS) analysis to identify and measure the perioperative differential metabolites in the plasma of adrenal pheochromocytoma patients. We identified differentially expressed genes by comparing the transcriptomic data of pheochromocytoma with the normal adrenal medulla. Through conducting two steps of metabolomics analysis, we identified 111 differential metabolites between the healthy group and the patient group, among which 53 metabolites were validated. By integrating the information of differential metabolites and differentially expressed genes, we inferred that the cysteine-methionine, pyrimidine, and tyrosine metabolism pathways were the three main metabolic pathways altered by the neoplasm. The analysis of transcription levels revealed that the tyrosine and cysteine-methionine metabolism pathways were downregulated in pheochromocytoma, whereas the pyrimidine pathway showed no significant difference. Finally, we developed an optimized diagnostic model of two metabolites, L-dihydroorotic acid and vanylglycol. Our results for these metabolites suggest that they may serve as potential clinical biomarkers and can be used to supplement and improve the diagnosis of pheochromocytoma.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Germination Increases the Glucomoringin Content in Moringa Sprouts via Transforming Tyrosine.

Moringa seeds are an excellent dietary source of phytochemicals (i.e., glucosinolates, GSLs; isothiocyanates, ITCs) with health-beneficial effects. Although numerous studies have been conducted on moringa seeds, the effect of germination on the regulation of GSLs remains scarcely explored. The present study investigated the dynamic changes of GSLs in moringa seeds during germination (at 25, 30, and 35 °C for 6 days in the dark) through an untargeted metabolomics approach and compared the antioxidant capacity of ungerminated and germinated moringa seeds. Our results showed that germination significantly increased the total GSL content from 150 (day 0) to 323 µmol/g (35 °C, day 6) on a dry weight (DW) basis, especially glucomoringin (GMG), the unique glucosinolate in moringa seeds, which was significantly upregulated from 61 (day 0) to 149 µmol/g DW (35 °C, day 4). The upregulation of GMG corresponded to the metabolism of tyrosine, which might be the initial precursor for the formation of GMG. In addition, germination enhanced the total ITC content from 85 (day 0) to 239 µmol SE/g DW (35 °C, day 6), indicating that germination may have also increased the activity of myrosinase. Furthermore, germination remarkably increased the total phenolic content (109-507 mg GAE/100 g DW) and antioxidant capacity of moringa seeds. Our findings suggest that moringa sprouts could be promoted as a novel food and/or ingredient rich in GMG.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Self-Assembling Triple-Helix Recombinant Collagen Hydrogel Enriched with Tyrosine.

The self-assembly of collagen within the human body creates a complex 3D fibrous network, providing structural integrity and mechanical strength to connective tissues. Recombinant collagen plays a pivotal role in the realm of biomimetic natural collagen. However, almost all of the reported recombinant collagens lack the capability of self-assembly, severely hindering their application in tissue engineering and regenerative medicine. Herein, we have for the first time constructed a series of self-assembling tyrosine-rich triple helix recombinant collagens, mimicking the structure and functionality of natural collagen. The recombinant collagen consists of a central triple-helical domain characterized by the (Gly-Xaa-Yaa)n sequence, along with N-terminal and C-terminal domains featuring the GYY sequence. The introduction of GYY has a negligible impact on the stability of the triple-helical structure of recombinant collagen while simultaneously promoting its self-assembly into fibers. In the presence of [Ru(bpy)3]Cl2 and APS as catalysts, tyrosine residues in the recombinant collagen undergo covalent cross-linking, resulting in a hydrogel with exceptional mechanical properties. The recombinant collagen hydrogel exhibits outstanding biocompatibility and bioactivity, significantly enhancing the proliferation, adhesion, migration, and differentiation of HFF-1 cells. This innovative self-assembled triple-helix recombinant collagen demonstrates significant potential in the fields of tissue engineering and medical materials.

Hydrogen Sulfide Exerted a Pro-Angiogenic Role by Promoting the Phosphorylation of VEGFR2 at Tyr797 and Ser799 Sites in Hypoxia-Reoxygenation Injury.

The protective effects of hydrogen sulfide (H2S) against ischemic brain injury and its role in promoting angiogenesis have been established. However, the specific mechanism underlying these effects remains unclear. This study is designed to investigate the regulatory impact and mechanism of H2S on VEGFR2 phosphorylation. Following expression and purification, the recombinant His-VEGFR2 protein was subjected to LC-PRM/MS analysis to identify the phosphorylation sites of VEGFR2 upon NaHS treatment. Adenovirus infection was used to transfect primary rat brain artery endothelial cells (BAECs) with the Ad-VEGFR2WT, Ad-VEGFR2Y797F, and Ad-VEGFR2S799A plasmids. The expression of VEGFR2 and recombinant Flag-VEGFR2, along with Akt phosphorylation, cell proliferation, and LDH levels, was assessed. The migratory capacity and tube-forming potential of BAECs were assessed using wound healing, transwell, and tube formation assays. NaHS notably enhanced the phosphorylation of VEGFR2 at Tyr797 and Ser799 sites. These phosphorylation sites were identified as crucial for mediating the protective effects of NaHS against hypoxia-reoxygenation (H/R) injury. NaHS significantly enhanced the Akt phosphorylation, migratory capacity, and tube formation of BAECs and upregulated the expression of VEGFR2 and recombinant proteins. These findings suggest that Tyr797 and Ser799 sites of VEGFR2 serve as crucial mediators of H2S-induced pro-angiogenic effects and protection against H/R injury.

ß-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells.

ß-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular ß-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of ß-catenin, suggestive of a Nek10 role in the control of ß-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for ß-catenin degradation, where it phosphorylates ß-catenin at Tyr30, located within the regulatory region governing ß-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of ß-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.

Integrated metabolomic and transcriptomic analyses provide insights into regulation mechanisms during bulbous stem development in the Chinese medicinal herb plant, Stephania kwangsiensis.

BACKGROUND: Stephania kwangsiensis Lo (Menispermaceae) is a well-known Chinese herbal medicine, and its bulbous stems are used medicinally. The storage stem of S. kwangsiensis originated from the hypocotyls. To date, there are no reports on the growth and development of S. kwangsiensis storage stems. RESULTS: The bulbous stem of S. kwangsiensis, the starch diameter was larger at the stable expanding stage (S3T) than at the unexpanded stage (S1T) or the rapidly expanding stage (S2T) at the three different time points. We used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Illumina sequencing to identify key genes involved in bulbous stem development. A large number of differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were identified. Based on the differential expression profiles of the metabolites, alkaloids, lipids, and phenolic acids were the top three differentially expressed classes. Compared with S2T, significant changes in plant signal transduction and isoquinoline alkaloid biosynthesis pathways occurred at both the transcriptional and metabolic levels in S1T. In S2T compared with S3T, several metabolites involved in tyrosine metabolism were decreased. Temporal analysis of S1T to S3T indicated the downregulation of phenylpropanoid biosynthesis, including lignin biosynthesis. The annotation of key pathways showed an up-down trend for genes and metabolites involved in isoquinoline alkaloid biosynthesis, whereas phenylpropanoid biosynthesis was not completely consistent. CONCLUSIONS: Downregulation of the phenylpropanoid biosynthesis pathway may be the result of carbon flow into alkaloid synthesis and storage of lipids and starch during the development of S. kwangsiensis bulbous stems. A decrease in the number of metabolites involved in tyrosine metabolism may also lead to a decrease in the upstream substrates of phenylpropane biosynthesis. Downregulation of lignin synthesis during phenylpropanoid biosynthesis may loosen restrictions on bulbous stem expansion. This study provides the first comprehensive analysis of the metabolome and transcriptome profiles of S. kwangsiensis bulbous stems. These data provide guidance for the cultivation, breeding, and harvesting of S. kwangsiensis.

Generation of Broad Protection against Influenza with Di-Tyrosine-Cross-Linked M2e Nanoclusters.

Tyrosine cross-linking has recently been used to produce nanoclusters (NCs) from peptides to enhance their immunogenicity. In this study, NCs were generated using the ectodomain of the ion channel Matrix 2 (M2e) protein, a conserved influenza surface antigen. The NCs were administered via intranasal (IN) or intramuscular (IM) routes in a mouse model in a prime-boost regimen in the presence of the adjuvant CpG. After boost, a significant increase in anti-M2e IgG and its subtypes was observed in the serum and lungs of mice vaccinated through the IM and IN routes; however, significant enhancement in anti-M2e IgA in lungs was observed only in the IN group. Analysis of cytokine concentrations in stimulated splenocyte cultures indicated a Th1/Th17-biased response. Mice were challenged with a lethal dose of A/California/07/2009 (H1N1pdm), A/Puerto Rico/08/1934 (H1N1), or A/Hong Kong/08/1968 (H3N2) strains. Mice that received M2e NCs + CpG were significantly protected against these strains and showed decreased lung viral titers compared with the naive mice and M2e NC-alone groups. The IN-vaccinated group showed superior protection against the H3N2 strain as compared to the IM group. This research extends our earlier efforts involving the tyrosine-based cross-linking method and highlights the potential of this technology in enhancing the immunogenicity of short peptide immunogens.

Biosynthesis of Hesperetin, Homoeriodictyol, and Homohesperetin in a Transcriptomics-Driven Engineered Strain of Streptomyces albidoflavus.

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus, we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus, we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Traditional medicinal use is linked with apparency, not specialized metabolite profiles in the order Caryophyllales.

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.

Combining fecal 16 S rRNA sequencing and spinal cord metabolomics analysis to explain the modulatory effect of PPARα on neuropathic pain.

BACKGROUND: Existing evidence suggests that the composition of the gut microbiota is associated with neuropathic pain (NP), but the mechanistic link is elusive. Peroxisome proliferator-activated receptor α (PPARα) has been shown to be a pharmacological target for the treatment of metabolic disorders, and its expression is also involved in inflammatory regulation. The aim of this study was to investigate the important modulatory effects of PPARα on gut microbiota and spinal cord metabolites in mice subjected to chronic constriction injury. METHODS: We analyzed fecal microbiota and spinal cord metabolic alterations in mice from the sham, CCI, GW7647 (PPARα agonist) and GW6471 (PPARα antagonist) groups by 16 S rRNA amplicon sequencing and untargeted metabolomics analysis. On this basis, the intestinal microbiota and metabolites that were significantly altered between treatment groups were analyzed in a combined multiomics analysis. We also investigated the effect of PPARα on the polarization fractionation of spinal microglia. RESULTS: PPARα agonist significantly reduce paw withdrawal threshold and paw withdrawal thermal latency, while PPARα antagonist significantly increase paw withdrawal threshold and paw withdrawal thermal latency. 16 S rRNA gene sequencing showed that intraperitoneal injection of GW7647 or GW6471 significantly altered the abundance, homogeneity and composition of the gut microbiome. Analysis of the spinal cord metabolome showed that the levels of spinal cord metabolites were shifted after exposure to GW7647 or GW6471. Alterations in the composition of gut microbiota were significantly associated with the abundance of various spinal cord metabolites. The abundance of Licheniformes showed a significant positive correlation with nicotinamide, benzimidazole, eicosanoids, and pyridine abundance. Immunofluorescence results showed that intraperitoneal injection of GW7647 or GW6471 altered microglial activation and polarization levels. CONCLUSION: Our study shows that PPARα can promote M2-type microglia polarization, as well as alter gut microbiota and metabolites in CCI mice. This study enhances our understanding of the mechanism of PPARα in the treatment of neuropathic pain.

Citric Acid Controls the Activity of YopH Bacterial Tyrosine Phosphatase.

Purpose: Citric acid (CA) is a tricarboxylic acid with antioxidant and antimicrobial properties. Based on previous studies, the small compound with its three carboxylic groups can be considered a protein tyrosine phosphatase inhibitor. YopH, a protein tyrosine phosphatase, is an essential virulence factor in Yersinia bacteria. Materials and Methods: We performed enzymatic activity assays of YopH phosphatase after treatment with citric acid in comparison with the inhibitory compound trimesic acid, which has a similar structure. We also measured the cytotoxicity of these compounds in Jurkat T E6.1 and macrophage J774.2 cell lines. We performed molecular docking analysis of the binding of citric acid molecules to YopH phosphatase. Results: Citric acid and trimesic acid reversibly reduced the activity of YopH enzyme and decreased the viability of Jurkat and macrophage cell lines. Importantly, these two compounds showed greater inhibitory properties against bacterial YopH activity than against human CD45 phosphatase activity. Molecular docking simulations confirmed that citric acid could bind to YopH phosphatase. Conclusion: Citric acid, a known antioxidant, can be considered an inhibitor of bacterial phosphatases.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

Mechanistic insights into G-protein activation via phosphorylation mediated non-canonical pathway.

Activation of heterotrimeric G-proteins (Gαßγ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of Gα that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce Gα activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gαi3, a subtype of Gα-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. In addition to weakened salt bridges at the domain interface, phosphorylations induced the unfolding of helix αF, which contributed to increased flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gαi3. Although the larger domain separation in the phosphorylated system provided an unobstructed path for the nucleotide, the accelerated release of GDP was attributed to increased fluctuations in several conserved regions like P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

Protein feature engineering framework for AMPylation site prediction.

AMPylation is a biologically significant yet understudied post-translational modification where an adenosine monophosphate (AMP) group is added to Tyrosine and Threonine residues primarily. While recent work has illuminated the prevalence and functional impacts of AMPylation, experimental identification of AMPylation sites remains challenging. Computational prediction techniques provide a faster alternative approach. The predictive performance of machine learning models is highly dependent on the features used to represent the raw amino acid sequences. In this work, we introduce a novel feature extraction pipeline to encode the key properties relevant to AMPylation site prediction. We utilize a recently published dataset of curated AMPylation sites to develop our feature generation framework. We demonstrate the utility of our extracted features by training various machine learning classifiers, on various numerical representations of the raw sequences extracted with the help of our framework. Tenfold cross-validation is used to evaluate the model's capability to distinguish between AMPylated and non-AMPylated sites. The top-performing set of features extracted achieved MCC score of 0.58, Accuracy of 0.8, AUC-ROC of 0.85 and F1 score of 0.73. Further, we elucidate the behaviour of the model on the set of features consisting of monogram and bigram counts for various representations using SHapley Additive exPlanations.

Multiomics Analysis of PCB126's Effect on a Mouse Chronic-Binge Alcohol Feeding Model.

BACKGROUND: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE: To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS: Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS: Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION: Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.