Mutation Of Lysine Research Articles

How arginine inhibits substrate-binding domain 2 elucidated using molecular dynamics simulations.

The substrate-binding domain 2 (SBD2) is an important part of the bacterial glutamine (GLN) transporter and mediates binding and delivery of GLN to the transporter translocation subunit. The SBD2 consists of two domains, D1 and D2, that bind GLN in the space between domains in a closed structure. In the absence of ligand, the SBD2 adopts an open conformation with larger space between domains. The GLN binding and closing are essential for the subsequent transport into the cell. Arginine (ARG) can also bind to SBD2 but does not induce closing and inhibits GLN transport. We use atomistic molecular dynamics (MD) simulations in explicit solvent to study ARG binding in the presence of the open SBD2 structure and observed reversible binding to the native GLN binding site with similar contacts but no transition to a closed SBD2 state. Absolute binding free energy simulations predict a considerable binding affinity of ARG and GLN to the binding site on the D1 domain. Free energy simulations to induce subsequent closing revealed a strong free energy penalty in case of ARG binding in contrast to GLN binding that favors the closed SBD2 state but still retains a free energy barrier for closing. The simulations allowed the identification of the molecular origin of the closing penalty in case of bound ARG and suggested a mutation of lysine at position 373 to alanine that strongly reduced the penalty and allowed closing even in the presence of bound ARG. The study offers an explanation of the molecular mechanism of how ARG competitively inhibits GLN from binding to SBD2 and from triggering the transition to a closed conformation. The proposed Lys373Ala mutation shows promise as a potential tool to validate whether a conformational mismatch between open SBD2 and the translocator is responsible for preventing ARG uptake to the cell.

Abstract LB173: Discovery of ALTA-2618, the first allosteric, mutant-selective targeted therapy for AKT1 E17K driven cancers

Abstract BACKGROUND: AKT1 E17K is a clinically validated oncogenic driver mutated in ~1-2% of all cancers and is enriched in several solid tumors including breast (~5%), endometrial (~3%) and prostate (~1.5%) cancer. Over half of AKT1 E17K mutant breast and gynecologic cancers are homozygous for the mutant oncogene with loss of the wildtype (WT) AKT1 allele via copy-neutral loss-of-heterozygosity (CN-LOH), suggesting uniquely potent oncogene addiction in these tumor types. While inhibitors of WT PI3K or AKT have demonstrated clinical efficacy in HR+/HER2- metastatic breast cancers with PI3K/AKT pathway mutations, on-target adverse events - most commonly hyperglycemia, diarrhea, and rash - have hindered the tolerability and durability of these agents. We aimed to exploit the mutant lysine of the E17K variant to develop a covalent, highly mutant-selective inhibitor that would avoid the toxicities associated with WT AKT inhibition. METHODS: Structure-based design was guided by proprietary co-crystal structures, enabling a refined pharmacophore consensus model, which fueled novel small molecule covalent design iterations using an in-house computational chemistry cluster. Lead molecules were identified from cellular signaling assays assessing pAKT S473 suppression and selectivity over WT AKT1 and AKT2. Rapid PK assessments were performed in mice, rats, dogs, and cynomolgus monkeys. Molecules with low clearance and high oral bioavailability were advanced directly to breast and endometrial PDX models driven by AKT1 E17K mutations. RESULTS: We identified ALTA-2618, a potent (7nM EC50) inhibitor of AKT1 E17K with 22x selectivity over WT AKT1 and 140x selectivity over WT AKT2. ALTA-2618 and related analogues demonstrated covalent attachment to the AKT1 E17K protein via intact mass spectrometry, and electron density analysis of co-crystal structures of highly similar family members of ALTA-2618 reveals unambiguous covalent attachment to the K17 side chain. Based on favorable exposure-response relationships and ideal cross-species PK, ALTA-2618 was tested in once daily oral dosing and induced tumor regressions in AKT1 E17K mutant HR+/HER2low breast cancer, TNBC, and endometrial cancer PDXs at doses as low as 10 mg/kg/d. Complete responses were observed with 30 mg/kg/d in the HR+/HER2low breast PDX model that were sustained for 60 days of dosing without significant body weight loss or hyperglycemia. CONCLUSIONS: ALTA-2618 is a potent, highly mutant-selective AKT1 E17K inhibitor that demonstrates favorable PK and tolerability with oral once-daily dosing that enables prolonged tumor regressions in multiple AKT1 E17K-mutant PDX models. These data support a mid-2024 IND submission to investigate ALTA-2618 in patients with AKT1 E17K mutant cancers. Citation Format: Eric A. Murphy, Michael D. Bartberger, Maureen Ibanez, Troy T. Smith, Nicole Alvarez, Noel Timple, Xuefeng Zhu, Kevin Fan, Kevin Yu. Discovery of ALTA-2618, the first allosteric, mutant-selective targeted therapy for AKT1 E17K driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB173.

Regulation of urea cycle by reversible high-stoichiometry lysine succinylation.

The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.

Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification

Creating long-lasting memories requires learning-induced changes in gene expression, which are impacted by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histones are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies implicate histone variants as novel regulators of memory, effects of PTMs on the function of histone variants are rarely considered. We previously showed that the histone variant H2A.Z suppresses memory, but it is unclear if this role is impacted by H2A.Z acetylation, a PTM that is typically associated with positive effects on transcription and memory. To answer this question, we used a mutation approach to manipulate acetylation on H2A.Z without impacting acetylation of other histone types. Specifically, we used adeno-associated virus (AAV) constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or H2A.Z.1 with impaired acetylation (acetyl-defective) by replacing the same lysines with alanine (KA). Expressing the H2A.Z.1 acetyl-mimic (H2A.Z.1KQ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1KA preferentially impacts genes involved in synaptic function, suggesting that acetyl-defective H2A.Z.1 impairs memory by altering synaptic regulation. Finally, we describe, for the first time, that H2A.Z is also involved in alternative splicing of neuronal genes, whereby H2A.Z depletion, as well as expression of H2A.Z.1 lysine mutants influence transcription and splicing of different gene targets, suggesting that H2A.Z.1 can impact behavior through effects on both splicing and gene expression. This is the first study to demonstrate that direct manipulation of H2A.Z post-translational modifications regulates memory, whereby acetylation adds another regulatory layer by which histone variants can fine tune higher brain functions through effects on gene expression and splicing.

Succinylated acetyl-CoA carboxylase contributes to aflatoxin biosynthesis, morphology development, and pathogenicity in Aspergillus flavus

Acetyl-CoA carboxylase (ACC), which catalyzes acetyl-CoA to produce malonyl-CoA, is crucial for the synthesis of mycotoxins, ergosterol, and fatty acids in various genera. However, its biofunction in Aspergillus flavus has not been reported. In this study, the accA gene was deleted and site-mutated to explore the influence of ACC on sporulation, sclerotium formation, and aflatoxin B1 (AFB1) biosynthesis. The results revealed that ACC positively regulated conidiation and sclerotium formation, but negatively regulated AFB1 production. In addition, we found that ACC is a succinylated protein, and mutation of lysine at position 990 of ACC to glutamic acid or arginine (accAK990E or accAK990R) changed the succinylation level of ACC. The accAK990E and accAK990R mutations (to imitate the succinylation and desuccinylation at K990 of ACC, respectively) downregulated fungal conidiation and sclerotium formation while increasing AFB1 production, revealing that the K990 is an important site for ACC's biofunction. These results provide valuable perspectives for future mechanism studies of the emerging roles of succinylated ACC in the regulation of the A. flavus phenotype, which is advantageous for the prevention and control of A. flavus hazards.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted renal cancer cell proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote renal cancer cell proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in RCC patients positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease

In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.

Signal regulatory protein beta 2 is a novel positive regulator of innate anticancer immunity.

In recent years, the therapeutic (re)activation of innate anticancer immunity has gained prominence, with therapeutic blocking of the interaction of Signal Regulatory Protein (SIRP)-α with its ligand CD47 yielding complete responses in refractory and relapsed B cell lymphoma patients. SIRP-α has as crucial inhibitory role on phagocytes, with e.g., its aberrant activation enabling the escape of cancer cells from immune surveillance. SIRP-α belongs to a family of paired receptors comprised of not only immune-inhibitory, but also putative immune-stimulatory receptors. Here, we report that an as yet uninvestigated SIRP family member, SIRP-beta 2 (SIRP-ß2), is strongly expressed under normal physiological conditions in macrophages and granulocytes at protein level. Endogenous expression of SIRP-ß2 on granulocytes correlated with trogocytosis of cancer cells. Further, ectopic expression of SIRP-ß2 stimulated macrophage adhesion, differentiation and cancer cell phagocytosis as well as potentiated macrophage-mediated activation of T cell Receptor-specific T cell activation. SIRP-ß2 recruited the immune activating adaptor protein DAP12 to positively regulate innate immunity, with the charged lysine 202 of SIRP-ß2 being responsible for interaction with DAP12. Mutation of lysine 202 to leucine lead to a complete loss of the increased adhesion and phagocytosis. In conclusion, SIRP-ß2 is a novel positive regulator of innate anticancer immunity and a potential costimulatory target for innate immunotherapy.

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ Tcells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ Tcells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

The α1 integrin cytoplasmic tail interacts with phosphoinositides and interferes with Akt activation

Integrin α1β1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1β1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1β1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1β1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.

CoCl2 -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.

SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD+ -dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2 -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl2 -induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.

Effects of methylation of arginine residue 83 on the enzymatic activity of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase is an enzyme responsible for arsenic methylation, and it requires S-adenosyl-methionine (SAM) as a coenzyme. We here generated two mutants to clarify the role of the highly conserved 83rd arginine residue (Arg83) in Motif I, the SAM-binding domain, of human AS3MT. When the AS3MT activity was compared between the mutants and the wild type (WT) recombinant protein, little activity was detected in the glycine mutant (Arg83Gly) or lysine mutant (Arg83Lys). When we examined the ability of transfected HEK293 cells exposed to arsenite to methylate arsenic, the methylation ability was significantly reduced in Arg83Gly compared to the WT, but was not significantly different between Arg83Lys and WT. Western blot analysis of the recombinant WT and Arg83Gly with an antibody that recognizes methylated Arg showed that an Arg residue in the WT was mono- and di-methylated, but not in Arg83Gly. Furthermore, a peptide containing dimethylated Arg83 was detected by MALDI-TOF/MS of the WT digested with chymotrypsin. These results indicate that AS3MT maintains its enzymatic activity through the methyl modification of Arg83.

NSD2 methylates AROS to promote SIRT1 activation and regulates fatty acid metabolism-mediated cancer radiotherapy

Fatty acid metabolism plays a critical role in both tumorigenesis and cancer radiotherapy. However, the regulatory mechanism of fatty acid metabolism has not been fully elucidated. NSD2, a histone methyltransferase that catalyzes di-methylation of histone H3 at lysine 36, has been shown to play an essential role in tumorigenesis and cancer progression. Here, we show that NSD2 promotes fatty acid oxidation (FAO) by methylating AROS (active regulator of SIRT1) at lysine 27, facilitating the physical interaction between AROS and SIRT1. The mutation of lysine 27 to arginine weakens the interaction between AROS and SIRT1 and impairs AROS-SIRT1-mediated FAO. Additionally, we examine the effect of NSD2 inhibition on radiotherapy efficacy and find an enhanced effectiveness of radiotherapy. Together, our findings identify a NSD2-dependent methylation regulation pattern of the AROS-SIRT1 axis, suggesting that NSD2 inhibition may be a potential adjunct for tumor radiotherapy.

ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer by promoting USP15-mediated GPX2 stabilization

BackgroundMultitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC.MethodsRNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc.ResultsRNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the β-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1.Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282–503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC.ConclusionsOur results demonstrate that the Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.

Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains.

The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined lengths and connectivity. While in vitro enzymatic assembly using expressed E1-activating and E2-conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48- and K63-biased E2-conjugating enzymes and two new carbamate protecting groups-pyridoxal 5'-phosphate (PLP)-cleavable aminobutanamide carbamate (Abac group) and periodate-cleavable aminobutanol carbamate (Aboc group)-provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers by buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His-tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2.

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

352-OR: Regulation of Melanocortin Receptor 4 (MC4R) Ciliary Trafficking

The primary cilium, a hair-like projection at the surface of nearly every cell, functions as a signaling antenna in processes as diverse as vision and body weight homeostasis. Obesity is a cardinal symptom of many ciliopathies including Bardet-Biedl Syndrome (BBS). While we are beginning to understand how cilia participate in body weight homeostasis, the molecular pathway that leads to obesity in BBS patients remains unclear. We now know that the melanocortin receptor 4, a GPCR that regulates feeding behavior, localizes to cilia in hypothalamic neurons. Meanwhile, most BBS proteins assemble or regulate the BBSome, a protein complex that removes activated signaling receptors from cilia. To characterize the stimuli and mechanisms that regulate MC4R trafficking in and out of cilia, we recapitulated MC4R trafficking in ciliated cell lines amenable to rapid manipulation. Surprisingly, levels of MC4R in cilia remain under the detection limit in most cells unless treated with an inverse agonist of MC4R. In addition, interference with BBSome function also increases ciliary MC4R levels in all cells. Thus, the tonic activity of MC4R promotes the constitutive clearance of MC4R from cilia via BBSome-mediated exit. These results suggest that artificially increasing the ciliary levels of MC4R may augment its signaling activity. To selectively recognize activated GPCRs, the BBSome relies on specific ubiquitin chains linked at lysine 63 (K63Ub chains) that become attached onto activated GPCRs via β-arrestin-associated ubiquitin ligases. Depletion of β-arrestin blocked the constitutive exit of MC4R from cilia and mutation of ubiquitin acceptor lysines from MC4R and MRAP2 results in a measurable increase in the ciliary levels of MC4R. In addition, targeting a protease that specifically cleaves K63Ub chains to cilia completely blocks the exit of MC4R from cilia. We conclude that the tonically active MC4R is ubiquitinated in cilia and actively removed by the BBSome. Disclosure M.Nachury: None. Funding American Diabetes Association (1-20-VSN-03)

Src family kinases engage differential pathways for encapsulation into extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

Lysine acetylation regulates mitochondrial pyruvate carrier activity and cardiac pyruvate oxidation

Background/Objective: The normal heart can acutely switch fuel sources based on delivery. While this is beneficial in physiological scenarios, this flexibility is often lost in cardiac pathologies. During fasting, blood glucose levels drop and increased free fatty acid delivery causes the heart to shut off glucose/pyruvate oxidation and increase fat oxidation. This balance, known as the Randle cycle, has mainly been attributed to regulation of pyruvate dehydrogenase (PDH) activity via PDH phosphorylation. Hypothesis: Decreased pyruvate transport into the mitochondria via posttranslational modifications of the mitochondrial pyruvate carrier (MPC) plays a role in regulating cardiac pyruvate oxidation. Methods: Wildtype mice were allowed to consume normal chow ad libitum or were fasted for 24 hours. After euthanasia, plasma and hearts were collected and left-ventricular muscle fibers were saponin permeabilized and mitochondrial respiration measured in an Oroboros Oxygraph O2k. Cardiac protein lysates were immunoprecipitated with anti-acetyl-lysine beads, and western blotted using standard procedures. A bioluminescent resonance transfer (BRET) assay to detect conformational changes caused by pyruvate/inhibitor binding the MPC was used, and lysine mutant MPC2 constructs were generated by site-directed mutagenesis. Lastly, MPC2-/- H9C2 cardiomyocytes were generated by CRISPR, and respiration measured by Seahorse bioanalyzer in these cells after transfection with WT or lysine mutant MPC2 constructs. Results: 24h fasting reduced blood glucose and insulin concentrations, and increased circulating free fatty acids. Cardiac mitochondrial oxidation of pyruvate/malate was decreased, while oxidation of fatty acids (palmitoyl-CoA+carnitine) was increased in fasted hearts. Western blotting of cardiac lysates showed the expected increase in phosphorylation of PDH-E1α in fasted hearts, known to decrease PDH activity. Immunoprecipitating cardiac lysates with anti-acetyl-lysine beads and blotting for MPC2 suggested increased acetylation of MPC2 in fasted hearts. Using a model structure of the MPC, we have recently proposed and validated lysine 49 (K49) of MPC2 as a critical pyruvate binding site within the MPC. To assess the possible importance of K49 acetylation we mutated K49 to glutamine to mimic acetylation and observed that this K49Q-MPC2 mutant was no longer able to bind pyruvate or competitive inhibitors in a MPC BRET assay. We next created MPC2 knockout H9C2 cardiomyocytes by CRISPR-Cas9 and observed complete loss of MPC2 and MPC1 expression in these cells which decreased pyruvate respiration compared to wildtype H9C2s. Overexpression of wildtype MPC1 and MPC2 constructs increased pyruvate respiration in these MPC-/- cells, while overexpression of the MPC2-K49Q mutant was unable to improve pyruvate respiration. Conclusion: Fasting results in decreased cardiac oxidation of pyruvate in part by acetylation of the MPC and decreased mitochondrial pyruvate transport. National Institutes of Health and SLU institutional funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of β-mercaptoethanol (β-ME), the abasic site unsaturated aldehyde forms a β-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.