This is the first theoretical investigation that systematically analyzes the interactions between the hexachlorophosphazene, [PCl2N]3, and small molecule impurities H2O and HCl in a 1 : 1 stoichiometric ratio. Utilizing both ab initio methods and seven DFT functionals in conjunction with the triple-ζ basis set, the pivotal structures in proposed reaction mechanisms are fully characterized and the energy change for each step was determined at the CCSD(T)/aTZ‖MP2/aTZ level of theory. Our QM calculations show that [PCl2N]3 can be hydrolyzed via a single-step mechanism with an activation energy of ca. 180 kJ mol-1, or be ring-opened by HCl through a two-step mechanism, in which the rate-determining step has an activation energy of ca. 120 kJ mol-1. Because the activation energy of these two reactions is notably lower than that of the ring-opening polymerization and the ring-ring expansion equilibrium (which requires ca. 240 kJ mol-1 of energy determined at a comparable DFT level of theory), our study indicates that even trace amount of H2O and HCl can significantly interfere with the polymerization process. Beyond revealing new mechanistic details, our calculations also indicate that all selected functionals can provide reasonable electronic structures to describe the reaction progress. On the other hand, while each of the functionals investigated here excels in closely matching the CCSD(T)/aTZ‖MP2/aTZ energy barriers for certain steps in the reaction, the B3LYP functional is capable of providing the most consistent results. This establishes that the B3LYP functional can be suitable for investigating phosphazene reactions as a computationally efficient and robust quantum mechanical approach while maintaining near-ab initio accuracy.

Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis. One of the hallmarks of TMAO’s role in CVD development is through alteration of cholesterol homeostasis, contributing to the development and progression of atherosclerosis. Current research investigating TMAO’s effects on cholesterol homeostasis focuses on the gut, liver, and vasculature. However, essentially nothing is known in regard to its impact on cholesterol balance in the adrenal gland, which is responsible for the synthesis of important steroid hormones derived solely from cholesterol, such as glucocorticoids and mineralocorticoids. With TMAO’s known effect on cholesterol homeostasis in other tissues, and the reliance of the adrenal cortex on lipoprotein-derived cholesterol to produce steroid hormones, it is important to further understand whether TMAO may be associated with altered adrenal activity, as conditions of adrenal dysfunction are often associated with increased risk for cardiovascular disease due to the role that glucocorticoids and mineralocorticoids play in the regulation of glucose metabolism and electrolyte homeostasis, respectively. Here, we performed a study on C57BL/6J mice to investigate whether changes in plasma TMAO levels resulted in altered transcriptomic expression of adrenocortical cholesterol transporters and steroidogenic enzymes. Six-week-old female C57BL/6J mice were placed on high fat diets, with half supplemented with gut bacterial choline TMA-lyase inhibitor, fluoromethylcholine (FMC) (n = 6). The mice fed ad libitum for a period of two weeks, after which adrenal glands were harvested. Real time polymerase chain reaction (qPCR) was used to evaluate transcriptomic expression of several adrenal cholesterol transporters and steroidogenesis enzymes. Results were analyzed using GraphPad Prism 10 student unpaired t-test function, where results with p values less than 0.05 were considered statistically significant. Three adrenocortical genes were found to be upregulated when mice were treated with FMC. mRNA expression of HDL-c transporter Srb1 trended higher (p = 0.057) in mice fed a diet supplemented with FMC, while Ldlr showed no trends or statistical significance. mRNA expression of cholesterol exporter Abca1 was significantly higher in mice fed a diet supplemented with FMC (p = 0.038), while Abcg1 showed no significance. Additionally, mRNA expression of steroidogenic enzymes Cyp11a1 and Cyp11b1 was significantly higher in mice treated with FMC compared to controls (p values of 0.010 and 0.029, respectively), where Cyp11a1 encodes the cholesterol side-chain cleavage enzyme (P450scc) and Cyp11b1 encodes the corticosterone synthase enzyme, 11B-hydroxylase. These results indicate that inhibition of TMAO-production does alter the transcription of critical genes involved in adrenal cholesterol flux and steroidogenesis. It appears that lack of TMAO results in moderate upregulation of an adrenal cholesterol transporter and two steroidogenic enzymes. Collectively, these data support a role for TMAO in adrenocortical cholesterol homeostasis and metabolism and merit further investigation into whether high plasma TMAO can impact adrenal function in a clinically significant way.

Phenylacetylglutamine (PAG) is a compound found in human circulation derived from the gut microbial metabolite phenylacetic acid. This compound has recently been identified through a collection of metabolomic studies and animal models to be both clinically and mechanistically linked to the development of cardiovascular disease. The multistep, metaorganismal production of PAG begins with dietary protein derived phenylalanine, which when taken up into gut microbes is converted to phenylpyruvic acid by multiple catalytic strategies. Phenylpyruvic acid is further metabolized through at least two distinctive microbial pathways. One pathway involves an oxidative decarboxylation by microbial phenylpyruvate ferredoxin:oxidoreductase (PPFOR), producing phenylacetyl-CoA, which is further hydrolyzed to phenylacetic acid. Alternatively, a non-oxidative decarboxylation reaction is catalyzed by phenylpyruvate decarboxylase (PPDC), producing phenylacetaldehyde, which is further oxidized to phenylacetic acid. Ultimately, phenylacetic acid, when taken up by the human host, is rapidly conjugated to glutamine to form phenylacetylglutamine by a phase II detoxification reaction for improved urinary excretion. Towards the aim of prevention and treatment of PAG-linked diseases we have developed gut microbe targeting, small molecule inhibitors designed to inhibit microbial phenylacetic acid production in order to suppress host PAG levels. We have synthesized a series of small molecule inhibitors designed to selectively target PPFOR and PPDC, taking advantage of the fact that while both enzymes perform distinct reactions they both share a common point by requiring thiamine pyrophosphate (TPP) as a cofactor for catalysis. These compounds are designed as mechanism-based inhibitors: substrate analogues, which upon catalysis, produce reactive electrophilic species capable of reacting with nucleophilic active site residues, irreversibly inactivating the target enzymes. This strategy seeks to create potent, highly selective inhibitors, due to dependency on the specific enzymatic reaction for inhibitor activation. To assay in vitro inhibitor efficacy, several distinct assays of microbial phenylacetic acid production were established using HPLC-MS/MS and spectrophotometric techniques (e.g. recombinant microbial enzymes cloned and expressed using bacterial expression vectors, and systems of monocultures or polymicrobial communities). For example, assays of recombinant PPDC were developed by cloning PPDC from the aerotolerant microbe P. mirabilis and quantifying the conversion of phenylpyruvic acid into phenylacetaldehyde by HPLC-MS/MS; assays of recombinant PPFOR were also developed (requiring strict anaerobic conditions) using recombinant PPFOR from the anaerobe B. thetaiotaomicron, and monitoring the CoA dependent oxidative decarboxylation of phenylpyruvic acid by spectrophotometric detection of the coupled reduction of methyl viologen. When tested in our assays, several of these compounds have shown potent inhibition (EC50 in the sub-micromolar range) of both recombinant PPFOR and PPDC as well as inhibition of phenylacetic acid production in polymicrobial cultures. Our ongoing studies are aimed at further refining structure-activity relationships (SAR) for lead candidates to optimize pre-specified PK-PD requirements, with the long term goal of utilizing these inhibitors in preclinical animal models of disease. Our results thus far demonstrate feasibility in creating single non-lethal high potency small molecule inhibitors for the potential therapeutic targeting of a gut-microbiome pathway that is clinically and mechanistically linked to the development and progression of cardiovascular disease – the PAG pathway.

An important microbial enzymatic pathway in the gut is choline/phosphatidylcholine degradation in which intestinal microbes utilize dietary choline to produce TMA via the choline utilization cluster polypeptide C (CutC) enzyme, a member of the glycyl radical enzyme family. Our goal in this theoretical work was to study the reaction mechanism and elucidate how the enzyme environment (steric and electronic) modulates the reaction path. Dissecting the effect of the enzyme environment on the reaction mechanism and shedding light on how steric and electronic effects affect the reaction path is an insightful and significant contribution of this work. Our theoretical results suggest that the final product of enzyme catalysis might be carbinolamine and not TMA and acetaldehyde. In addition, we found out that Glu491 plays the role of a base in this reaction (a disputed fact) by temporarily abstracting a proton from the hydroxylic group of choline sometime during the reaction—with the proton transfer being critical for the reaction to proceed to completion. We also found that the choice of computational protocol not only alters the reaction energetics but can change the reaction path by creating new intermediates and transition states or eliminating existing ones.

Quenching peroxynitrite (a reactive oxidant species) is a vital process in biological systems and environmental chemistry as it maintains redox balance and mitigates damaging effects in living cells and the environment. In this study, we report a systematic analysis of the mechanism of transforming peroxynitrite into nitrate using diaryl selenide in water. Through quantum mechanical calculations, we investigate the dynamic isomerization of peroxynitrite in a homogeneous catalytic environment. The mapped potential energy surfaces (PESs) generated using various methods in conjunction with different basis sets suggest that the isomerization mechanism includes four major steps: the reaction of peroxynitrite with diaryl selenide via oxygen-bound selenium; selenium oxidation in the presence of an appropriate oxidant; oxygen transfer; and ultimately, the generation of nitrate. The molecular orbital analysis suggests a substituent effect on the aromatic ring of diaryl selenide in this reaction. Changes in both molecular orbitals and electrostatic potential highlight the significance of the electron transfer step in ensuring the progression of this reaction.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser 473 -AKT + . EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Introduction: The Aster-C protein (encoded by the Gramd1c gene) is an endoplasmic reticulum (ER) resident protein that has been reported to transport cholesterol from the plasma membrane to the ER. Although there is a clear role for the closely-related Aster-B protein in cholesterol transport and downstream esterification in the adrenal gland, the specific role for Aster-C in cholesterol homeostasis is not well understood. Here, we have examined whole body cholesterol balance in mice globally lacking Aster-C under low or high dietary cholesterol conditions.Method: Age-matched Gramd1c +/+ and Gramd1c -/- mice were fed either low (0.02%, wt/wt) or high (0.2%, wt/wt) dietarycholesterol and levels of sterol-derived metabolites were assessed in the feces, liver, and plasma.Results: Compared to wild type controls (Gramd1c +/+) mice, mice lackingGramd1c (Gramd1c -/-) have no significant alterations in fecal, liver, or plasma cholesterol. Given the potential role for Aster C in modulating cholesterol metabolism in diverse tissues, we quantified levels of cholesterol metabolites such as bile acids, oxysterols, and steroid hormones. Compared to Gramd1c +/+ controls, Gramd1c -/- mice had modestly reduced levels of select bile acid species and elevated cortisol levels, only under low dietary cholesterol conditions. However, the vast majority of bile acids, oxysterols, and steroid hormones were unaltered in Gramd1c -/- mice. Bulk RNA sequencing in the liver showed that Gramd1c -/- mice did not exhibit alterations in sterol-sensitive genes, but instead showed altered expression of genes in major urinary protein and cytochrome P450 (CYP) families only under low dietary cholesterol conditions.Discussion: Collectively, these data indicate nominal effects of Aster-C on whole body cholesterol transport and metabolism under divergent dietary cholesterol conditions. These results strongly suggest that Aster-C alone is not sufficient to control whole body cholesterol balance, but can modestly impact circulating cortisol and bile acid levels when dietary cholesterol is limited.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing β1- versus β2-adrenergic receptors (β1AR and β2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of β2AR, but not β1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on β2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical β2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR. Allosteric modulation is crucial in metabolic regulation but unexplored in gut microbehost interactions. Here the authors show gut microbe-derived phenylacetylglutamine acts as a negative allosteric modulator of β2-adrenergic receptors, impacting heart function.

The most popular method to synthesize polychlorophosphazenes, the parent of a prominent class of inorganic polymers, is the ring-opening polymerization (ROP) of [PCl2N](3). In contrast to the accepted (S(N)1-initiated) ROP mechanism that begins with heterolytic P-Cl bond cleavage in [PCl2N](3), our quantum mechanical (QM) calculations suggest that the ROP can proceed through a S(N)2-like route in which one [PCl2N](3) can be attacked by a neighboring [PCl2N](3) and hence transform through a four-center transition state (4C PNPCl TS), yielding a cyclic chlorophosphazene with a linear tail, termed a "tadpole". Meanwhile, two [PCl2N](3) molecules can morph into [PCl2N](6) (RR expansion) through a different four-center transition state (4C PNPN TS) without the assistance of a bridging chlorine. As the activation energy of these processes follows the trend tadpole backbite < chain branching < ROP initiation <= RR initiation = RR expansion < chain propagation (all within 241.2 +/- 16 kJ/mol), the ROP and RR mechanisms compete toward product formation. Not only does our pioneering QM calculations unveil the pivotal role of the bridging chlorine in the S(N)2 mechanism, it also explains its effect on reactivity of [PCl2N](3) species, underscoring the significance of halogen substituents in modulating polymerization. By comprehensively examining the ROP, RR, linear propagation, and ring closure processes, we attempt to resolve long-standing queries in chlorophosphazene research, elucidating the wide variability in reaction pot products and the necessity of halogen substituents in specific processes. Thus, this work characterizes a variety of four-center transition states for the first time and introduces a novel mechanistic process for polymerization. Finally, our work provides an explanation of the existence of the chlorinated tadpole.

Bile acids (BAs) are cholesterol-derived compounds that regulate glucose, lipid, and energy metabolism. Despite their significance in glucose homeostasis, the association between specific BA molecular species and their synthetic pathways with diabetes mellitus (DM) is unclear. Here, we used a recently validated stable-isotope dilution high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify a panel of BAs in fasting plasma from subjects (n=2,145) and explored structural and genetic determinants of BAs linked to DM, insulin resistance and obesity. Multiple 12α-hydroxylated BAs were associated with DM [adjusted odds ratios (aORs):1.3-1.9 (all P<0.05)] and insulin resistance [aORs:1.3-2.2 (all P<0.05)]. Conversely, multiple 6α-hydroxylated BAs and isolithocholic acid (Iso-LCA) were inversely associated with DM and obesity [aORs:0.3-0.9 (all P<0.05)]. Genome-wide association studies (GWAS) revealed multiple genome-wide significant loci linked with nine of the 14 DM-associated BAs, including a locus for Iso-LCA (rs11866815). Mendelian randomization analyses showed genetically elevated DCA levels were causally associated with higher BMI, and Iso-LCA levels were causally associated with reduced BMI and DM risk. In conclusion, comprehensive large-scale quantitative mass spectrometry and genetics analyses show circulating levels of multiple structurally specific BAs, especially DCA and Iso-LCA, are clinically associated with and genetically linked to obesity and DM.

AbstractHypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.

Although the synthesis of chlorophosphazene polymers has been explored for more than 100 years, the shortest yet most illusive monomer, Cl3P═NH, has never been isolated and fully characterized. Here we investigate the formation of Cl3P═NH from PCl5 and NH3 in chlorobenzene through quantum mechanical calculations. The potential energy surface was mapped using the MP2 Hamiltonian in conjunction with Dunning's correlation-consistent basis sets (aug-cc-pVXZ, where X = D and T). Along with HOMO/LUMO frontier molecular orbitals and natural bond orbital analyses, we found that instead of following the SN1 path proposed in the literature, the reaction proceeds via an addition-elimination mechanism. Our results also indicate that due to the low-lying stable intermediates (IM), most steps are exothermic such that the production of Cl3P═NH·2HCl can be completed once the energy barrier for the formation of [PCl4-NH3]+Cl- is overcome. Therefore, our theoretical work might explain the challenges in isolating any of the IMs in a typical chlorophosphazene reaction in chlorobenzene.

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardio-vascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Us-ing genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferre-doxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decar-boxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln for-mation in vivo.

Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.

Background: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. Methods: After synthesizing [S-34]H2S and developing sample preparation protocols that avoid sulfide contami-nation with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glu-tamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. Results: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra-and inter-day reproducibility (<= 7.6%, and <= 12.7%, respectively), linearity (r(2) > 0.997), recovery (90.9%-110%) and stability (90.0%-100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 +/- 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0-66.7% reduction with antibiotics). Conclusions: A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.

In mammalian cells, 20 aminoacyl-tRNA synthetases (AARS) catalyze the ligation of amino acids to their cognate tRNAs to generate aminoacylated-tRNAs. In higher eukaryotes, 9 of the 20 AARSs, along with 3 auxiliary proteins, join to form the cytoplasmic multi-tRNA synthetase complex (MSC). The complex is absent in prokaryotes, but evolutionary expansion of MSC constituents, primarily by addition of novel interacting domains, facilitates formation of subcomplexes that join to establish the holo-MSC. In some cases, environmental cues direct the release of constituents from the MSC which enables the execution of non-canonical, i.e., "moonlighting", functions distinct from their essential activities in protein translation. These activities are generally beneficial, but can also be deleterious to the cell. Elucidation of the non-canonical activities of several AARSs residing in the MSC suggest they are potential therapeutic targets for cancer, as well as metabolic and neurologic diseases. Here, we describe the role of MSC-resident AARSs in cancer progression, and the factors that regulate their release from the MSC. Also, we highlight recent developments in therapeutic modalities that target MSC AARSs for cancer prevention and treatment.

Protein lysine carbamylation is an irreversible post translational modification resulting in generation of homocitrulline (N-epsilon-carbamyllysine), which no longer possesses a charged epsilon-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modifi- cation in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor " apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Despite recent advances in structural determination of individual proteins, elucidating the 3-dimensional architecture of large, multiprotein complexes remains challenging, partly because of issues related to structural integrity during purification. Here, we describe a protocol to determine the 3-dimensional architecture of the 11-constituent, multi-tRNA synthetase complex (MSC) using chemical cross-linking coupled with mass-spectrometry (XL-MS). The protocol does not require purification and is broadly applicable, facilitating determination of native structures in cell lysates and in non-disrupted cells as well as in purified complexes. For complete details on the use and execution of this protocol, please refer to Khan et al. (2020).

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)—essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a “multisite” mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second “piggy-back” mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

Cellular cholesterol is regulated by at least two transcriptional mechanisms involving sterol-regulatory-element-binding proteins (SREBPs) and liver X receptors (LXRs). Although SREBP and LXR pathways are the predominant mechanisms that sense cholesterol in the endoplasmic reticulum and nucleus to alter sterol-regulated gene expression, evidence suggests cholesterol in plasma membrane can be sensed by proteins in the Hedgehog (Hh) pathway which regulate organ self-renewal and are a morphogenic driver during embryonic development. Cholesterol interacts with the G-protein-coupled receptor Smoothened (Smo), which impacts downstream Hh signaling. Although evidence suggests cholesterol influences Hh signaling, it is not known whether Smo-dependent sterol sensing impacts cholesterol homeostasis in vivo. We examined dietary-cholesterol-induced reorganization of whole-body sterol and bile acid (BA) homeostasis in adult mice with inducible hepatocyte-specific Smo deletion. These studies demonstrate Smo in hepatocytes plays a regulatory role in sensing and feedback regulation of cholesterol balance driven by excess dietary cholesterol.