Atherosclerosis underlies most cardiovascular diseases and is driven by complex cellular and molecular interactions within the vessel wall. While mechanisms promoting lesion formation have been extensively studied, far less is known about pathways that regulate lesion regression and stabilization. O-GlcNAcylation is a nutrient-sensitive post-translational modification that alters protein function and cellular signaling. We previously reported that loss of O-GlcNAc transferase (OGT), a key regulator of O-GlcNAcylation, in smooth muscle cells (SMC) prevents hyperglycemia-induced atherosclerosis. However, whether loss of smooth muscle O-GlcNAcylation affects established lesions once formed remains unclear. To address this, we utilized Apoe-/- mice with conditional SMC-specific Ogt deletion, generated via tamoxifen (tmx)-inducible SMC-restricted Cre driver (Myh11-CreERT2). Five-week-old male Apoe-/-Cretg mice bearing Ogtfl/y or Ogt+/y were fed a Western diet for eight weeks to induce advanced atherosclerotic lesions. At 14-week age, Cre recombination was induced via tmx injection (40mg/kg/day x 5 days) to generate Apoe-/-smOgtKO and Apoe-/-smOgtWT mice. Both groups were then transitioned to standard lab chow for nine weeks to promote lesion regression. At 23-week-age, mice were harvested; aortic roots and aortic vessels were collected for further analysis. Aortic root sections were assessed for plaque size, lipid burden, and collagen content via histological staining and microscopy. In parallel, aortic tissue lysates were used for high-throughput proteomic profiling. Morphometric analysis revealed a significant increase in lipid burden (Oil red O) and collagen content (Masson’s Trichrome) of aortic root lesions in Apoe-/-smOgtKO vs. Apoe-/-smOgtWT mice, while there was no difference in plaque area (H & E) between these mice. Proteomic analysis revealed a total of 4632 proteins, out of which 135 differentially expressed proteins were identified in Apoe-/-smOgtKO aortic vessels, at |log 2 FC| ≥ 1 and p ≤ 0.05 cut-off values. Specifically, top upregulated genes included Chad, P2rx1, Postn (log2FC > 1.5, p < 0.003); top downregulated genes included Itga8, Eln, Epn2 (log2FC ≥ -1.48, p ≤ 0.004). Gene Ontology enrichment analysis of differentially abundant proteins using GO-BP, GO-MF, and GO-CC categories revealed negative enrichment for processes related to cell-cell adhesion and cell-extracellular matrix adhesion in Apoe-/-smOgtKO aortic vessels. Moreover, hallmark analysis revealed significant upregulation of PI3K, AKT, and MTOR signaling pathways, accompanied by suppression of myogenesis in Apoe-/-smOgtKO vessels, using |NES| ≥ ~1.5 and FDR ≤ 0.05 cut-off values. These findings indicate that loss of smooth muscle O-GlcNAcylation during lesion regression increases collagen deposition, coupled with dysregulation of adhesion-related signaling pathways. Overall, our data suggest a novel regulatory role of smooth muscle O-GlcNAcylation in vascular smooth muscle cell signaling and plaque stability, with implications for cardiovascular disease risk and therapy. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore are established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2, is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion, and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of the GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn't clear. This study demonstrated that the histone acetyl transferase p300, acts as a pivotal factor which on one hand causes acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down, and over-expression experiments. Furthermore, SP1 degradation, SMAD activation, and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator, and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

Abstract Background: clear cell Renal Cell Carcinoma (ccRCC), the predominant form of adult kidney cancer, is typically treated using Tyrosine Kinase Inhibitors (TKIs), Immune Checkpoint inhibitors (ICIs), and (recently) HIF2α blockade. Unfortunately, these therapies ultimately fail to cure advanced ccRCC in many patients. New targets that complement these therapies are thus urgently required. The Nuclear Factor IA (NF-I/A) transcription factor (TF) represents one such HIF-independent target in ccRCC. Methods: VHL loss globally alters the tumor epigenome. Using ChIP-Seq, we measured the changes in H3K27 acetylation (H3K27ac), a histone mark associated with gene activation. We custom-generated a CRISPR/Cas9 library targeting the top 400 genes marked by increased H3K27ac and higher gene expression (measured by RNA-Seq) in VHL-deficient cells. CRISPR/Cas9 dropout screens were performed in vitro and in vivo (6 biological replicates) and top candidates were identified using the STARS algorithm. Validation studies were done in a panel of ccRCC lines using in vitro assays (e.g., Cut-&-Run, qPCR, western blots, and proliferation) and in ccRCC mouse models in vivo. Triplicate data was analyzed and compared statistically using ANOVA or Student’s t-test, corrected for multiple testing (as needed); p<0.05 considered significant. NF-I/A’s interactome and post-translational modifications (PTMs) were analyzed using label-free quantitative Mass Spectrometry. Clinical validations were performed using publicly available TCGA datasets and additionally using the ccRCC patient cohorts at MSKCC and at the Yale School of Medicine. Results: Our in vitro and in vivo CRISPR/Cas9 screens identified the Nuclear Factor IA (NF-I/A) transcription factor (TF) as an oncogenic dependency in ccRCC. NF-I/A loss led to fitness defects in vitro and diminished tumor growth in vivo, both in localized and metastatic tumors. These effects were rescued with wild-type NF-I/A but not a TF activity dead mutant. Molecular analysis in cells and validations in human tumor datasets suggest that: (1) NF-I/A regulates adipogenic programs in lipid-rich ccRCCs; (2) Higher NF-I/A expression is associated with dysregulated angiogenic signatures and lower immune infiltration in human tumors; and (3) Lower NF-I/A expression was associated with better ICI response. Binding studies and drug screening approaches have identified NF-I/A PTMs and transcriptional complexes that might be amenable to future targeting. Conclusions: We discovered NF-I/A as a novel, HIF-independent, oncogenic dependency in kidney cancer. NF-I/A has been studied in metabolic disorders, but its cancer relevance is relatively unknown. We address this conceptual gap and demonstrate that NF-I/A’s adipogenic function governs tumor metabolism and alters the production of lipid-derived chemo-attractants (e.g., leukotrienes). Thus, NF-l/A functions as both a cell-intrinsic oncogene and a host immune modulator. Altogether, these findings establish the foundations for future drug discovery efforts to target NF-I/A’s oncogenic function. Citation Format: Carleigh Salem, Jason Scovell, Shannon Zayas, Cerise Tang, Anasuya Dighe, Treg Grubb, Neil Ruthen, Suza Mohammad Nur, Belinda Willard, Sakari Vanharanta, Fengshen Kuo, A Ari Hakimi, David Braun, Ed Reznik, Abhishek A. Chakraborty. Epigenetic Dysfunction Promotes Dependence on the NF-I/A Transcription Factor in Kidney Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr PR015.

Although strong evidence links the gut microbiome to metabolic disease, the mechanisms linking microbiota to hormonal and metabolic responses to food are not well understood. After a meal, gut bacteria produce a wide array of small molecule, protein, and lipid metabolites originating from bacterial sources. Attributing physiological function to select gut microbe-derived metabolites is critical to understanding diet-microbe-host interactions, and to developing microbiome-inspired therapies to improve human health. Here, we have investigated the role of a poorly annotated class of gut microbiome-derived lipids called N-acyl amides in postprandial metabolic physiology. Here, we show that both bacterial production and provision of exogenous N-acyl amides reorganize host hormone-driven metabolic transition after a meal. Moreover, N-acyl amides exert broad effects on the meal-and circadian-related reorganization of gene expression, metabolic hormones, and gut microbiome composition. Collectively, these results demonstrate that microbiota-derived N-acyl amides play a physiologic role in postprandial metabolic homeostasis in the host.

BACKGROUND: Dysregulated proteolysis is implicated in thoracic (thoracic aortic aneurysm [TAA]) and abdominal aortic aneurysm (AAA) pathogenesis, but proteolytic landscapes (degradomes) of aneurysmal and normal aorta and contributions of individual proteases remain undefined. Here, a proteome-wide approach was used to define and compare TAA and AAA degradomes and uncover the specific role in aortic remodeling of 2 proteases consistently identified in the aneurysms, CMA1 (mast cell chymase) and MMP9 (matrix metalloprotease 9). METHODS: The mass spectrometry-based N-terminomics strategy, terminal amine isotopic labeling of substrates, was applied to Marfan syndrome TAAs (n=5), AAAs (n=16), and nondiseased thoracic aorta (n=4), and abdominal aorta (n=4) in a forward degradomics application, that is, to define substrate and protease degradomes. 8-plex iTRAQ terminal amine isotopic labeling of substrates was used for quantitative comparison of the tissue cohorts. Cleavage sites of CMA1 and MMP9 were sought by reverse degradomics, that is, digestion of aortic proteins with these proteases, followed by terminal amine isotopic labeling of substrates. CMA1 and MMP9 proteolysis of biglycan was further resolved using amino-terminal oriented mass spectrometry of substrates. RESULTS: We experimentally annotated 20 885 proteolytically derived peptides and identified 129 proteases in the aortic tissues. Quantitative substrate degradome comparisons identified specific differentially modulated pathways and networks in TAAs and AAAs. Reverse degradomics elucidated >300 CMA1 and MMP9 substrate cleavage sites, of which many, including orthogonally validated biglycan cleavages, occurred in the disease degradomes. CONCLUSIONS: Unbiased forward degradomics of the aortic wall from TAA, AAA, and nondiseased tissue provides a systems biology view of aortic wall breakdown and a new resource for its hitherto occult proteolytic landscape, demonstrating widespread extracellular matrix remodeling with disproportionate impact on proteoglycans. The findings provided insights into aortic aneurysm pathways and disease biomarkers and suggest involvement of numerous proteases. Mapping of specific proteolytic contributions of CMA1 and MMP9 illustrates a strategy for defining the activities of all proteases involved in aortic disease.

Eph receptors, the largest subfamily of single-pass transmembrane receptor tyrosine kinases, play essential roles in development, including axon guidance and cell positioning, and in adult functions such as synaptogenesis. While their canonical signaling is typically repulsive and ligand-dependent, non-canonical activity promotes cell migration and proliferation in several cancers through ligand-independent mechanisms. Proteases associated with neurodegeneration can cleave Eph receptors, generating near full-length intracellular region (ICR) fragments, whose signaling potential remains unclear. Here, we dissect the mechanistic contributions of the sterile α motif (SAM) domain and tyrosine phosphorylation to the behavior of the EphA2 ICR-one of the best-characterized Eph receptors. The ICR expressed in E. coli retains kinase activity and undergoes phosphorylation at tyrosine residues identified in eukaryotic systems. This phosphorylation reduces domain interactions, as measured by microscale thermophoresis. AlphaFold2Multimer models and available crystal structures of EphA2 dimers provide limited mechanistic insights, likely due to crystal packing effects. Using coarse-grained molecular dynamics simulations (Martini 3.0), we find that SAM-kinase interactions are predominantly transient but show clustering in two kinase-domain regions. NMR spectroscopy with assigned EphA2 SAM domains reveals non-canonical ICR contacts, potentially keeping the canonical SAM interface accessible for partner binding. Introduction of the SHIP2 SAM domain results in canonical EphA2-SHIP2 interactions, along with unexpected contacts with the kinase domain. Together, our results highlight the dynamic, phosphorylation-sensitive nature of EphA2 ICR in solution and suggest a versatile signaling capacity for cleaved intracellular fragments in pathological contexts. Significance:Eph receptors are key signaling molecules in development and disease, yet the behavior of their intracellular region (ICR) following receptor cleavage remains poorly understood. This study reveals that the EphA2 ICR, including the sterile α motif (SAM) and kinase domains, adopt transient interactions between several configurations moderately affected by phosphorylation. Through a combination of biophysical assays, molecular dynamics simulations, and NMR spectroscopy, we identify regions of intra- and inter-domain contacts, including non-canonical interactions with partner SAM domains such as SHIP2. These findings provide critical insight into how cleaved EphA2 fragments remain functionally active in the cytoplasm and suggest a versatile, modular signaling capacity that may be relevant in pathological conditions like cancer and neurodegeneration.

BACKGROUND:Skeletal muscle is a major target for ethanol-induced perturbations, leading to sarcopenia in alcohol-related liver disease (ALD). The complex interactions and pathways involved in adaptive and maladaptive responses to ethanol in skeletal muscle are not well understood. Unlike hypothesis-driven experiments, an integrated multiomics-experimental validation approach provides a comprehensive view of these interactions. METHODS:We performed multiomics analyses with experimental validation to identify novel regulatory mechanisms of sarcopenia in ALD. Studies were done in a comprehensive array of models including ethanol-treated (ET) murine and human-induced pluripotent stem cell-derived myotubes (hiPSCm), skeletal muscle from a mouse model of ALD (mALD) and human patients with alcohol-related cirrhosis and controls. We generated 13 untargeted datasets, including chromatin accessibility (assay for transposase accessible chromatin), RNA sequencing, proteomics, phosphoproteomics, acetylomics and metabolomics, and conducted integrated multiomics analyses using UpSet plots and feature extraction. Key findings were validated using immunoblots, redox measurements (NAD+/NADH ratio), imaging and senescence-associated molecular phenotype (SAMP) assays. Mechanistic studies included mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX) to increase redox ratio and MitoTempo as a mitochondrial free radical scavenger. RESULTS:Multiomics analyses revealed enrichment in mitochondrial oxidative function, protein synthesis and senescence pathways consistent with the known effects of hypoxia-inducible factor 1α (HIF1α) during normoxia. Across preclinical and clinical models, HIF1α targets (n = 32 genes) and signalling genes (n > 100 genes) (n = 3 ATACseq, n = 65 phosphoproteomics, n = 10 acetylomics, n = 6 C2C12 proteomics, n = 106 C2C12 RNAseq, n = 64 hiPSC RNAseq, n = 30 hiPSC proteomics, n = 3 mouse proteomics, n = 25 mouse RNAseq, n = 8 human RNAseq, n = 3 human proteomics) were increased. Stabilization of HIF1α (C2C12, 6hEtOH 0.24 ± 0.09; p = 0.043; mALD 0.32 ± 0.074; p = 0.005; data shown as mean difference ± standard error mean) was accompanied by enrichment in the early transient and late change clusters, -log(p-value) = 1.5-3.8, of the HIF1α signalling pathway. Redox ratio was reduced in ET myotubes (C2C12: 15512 ± 872.1, p < 0.001) and mALD muscle, with decreased expression of electron transport chain components (CI-V, p < 0.05) and Sirt3 (C2C12: 0.067 ± 0.023, p = 0.025; mALD: 0.41 ± 0.12, p = 0.013). Acetylation of mitochondrial proteins was increased in both models (C2C12: 107364 ± 4558, p = 0.03; mALD: 40036 ± 18 987, p = 0.049). Ethanol-induced SAMP was observed across models (P16: C2C12: 0.2845 ± 0.1145, p < 0.05; hiPSCm: 0.2591, p = 0.041). MitoLbNOX treatment reversed redox imbalance, HIF1α stabilization, global acetylation and myostatin expression (p < 0.05). CONCLUSIONS:An integrated multiomics approach, combined with experimental validation, identifies HIF1α stabilization and accelerated post-mitotic senescence as novel mechanisms of sarcopenia in ALD. These findings show the complex molecular interactions leading to mitochondrial dysfunction and progressive sarcopenia in ALD.

The anti-apoptotic BCL2 family member MCL1 is overexpressed in many cancers and has been linked to chemoresistance. Unlike other BCL2 family members, MCL1 displays both well-defined mitochondrial anti-apoptotic activities and also emerging nuclear functions. Prior reports suggest that MCL1 enters the nucleus during chemotherapy and promotes chemoresistance by influencing cell cycle progression and DNA repair. These nuclear roles of MCL1, however, remain poorly characterized. Using a newly validated monoclonal antibody across several cell lines and treatments, we find no evidence that MCL1 enhances chemoresistance or preferentially accumulates in the nucleus after drug exposure. Proximity biotinylation identified novel nuclear MCL1 interactors but did not recover previously reported DNA repair or cell cycle partners. Thus, while MCL1 does reach the nucleus and interact with nuclear proteins, our data do not support a role for MCL1 in chemoresistance. Further work is needed to clarify the functional significance of nuclear MCL1.

Weill-Marchesani syndrome (WMS) is characterized by severe short stature, short hands and feet (brachydactyly), joint contractures, tight skin, and heart valve, eye, and skin anomalies. Whereas recessive WMS is caused by mutations in ADAMTS10, ADAMTS17, or LTBP2, dominant WMS is caused by mutations in FBN1 (encoding fibrillin-1). Since bone growth is driven by chondrocyte proliferation and hypertrophy in the growth plates, the genetics of WMS suggests that the affected ECM proteins act within the same pathway to regulate chondrocyte and growth plate function. Here, we investigated the role of the secreted ADAMTS proteases ADAMTS10 and ADAMTS17 in growth plate function and ECM formation. We generated Adamts10;Adamts17 double knockout (DKO) mice, which showed significant postnatal lethality compared to single Adamts10 or Adamts17 KO mice. Importantly, we observed severe bone shortening DKO mice, which correlated with a narrower hypertrophic zone in their growth plates. ADAMTS17 substrates identified by N-terminomics and yeast two-hybrid screening identified the ECM proteins fibronectin and collagen VI (COL6). However, validation experiments did not reveal direct proteolysis of either fibronectin or COL6 by ADAMTS17. We then investigated ECM formation in primary ADAMTS10- and ADAMTS17-deficient skin fibroblasts and observed compromised fibronectin deposition concomitant with aberrant intracellular accumulation of fibrillin-1. These findings support a role for ADAMTS17 in ECM protein secretion and assembly. Collectively, our data suggest that ADAMTS10 and ADAMTS17 regulate bone growth by regulating chondrocyte hypertrophy or hypertrophic chondrocyte turnover. Mechanistically, ADAMTS17 appears to be a critical regulator of ECM protein secretion or pericellular matrix assembly, whereas ADAMTS10 likely modulates ECM formation at later stages, possibly regulating the spatio-temporal deposition of fibrillin isoforms.

Introduction: Atrial fibrillation (AF) is a common cardiac arrhythmia. Putative genes associated with AF risk have been identified through large genome wide association studies. How expression of these proteins in the left atria differs by AF state (no history of AF, history of paroxysmal AF, history of persistent AF, persistent AF, long-standing persistent AF) has not been examined. Hypothesis: We hypothesized that proteins associated with AF risk differ by AF state. Methods: Left atrial appendage (LAA) tissue was obtained from 222 Cleveland Clinic patients undergoing cardiac surgery. At surgery, 104 were in sinus rhythm (SR) (24 without a history of AF, 50 had a history of paroxysmal AF, 30 had a history of persistent AF) and 118 patients were in AF rhythm (65 with history of persistent AF, 53 long-standing persistent AF). Proteins (n=2539) were identified by mass spectrometry. Protein levels were modeled for associations with AF state using linear regression and adjusted for sex, age, and 23 inferred surrogate variables. Results: Among the quantified proteins we identified 33 encoded from putative AF risk genes. Of these, CASQ2 was increased (p<0.05) and TTN was decreased in patients with a history of AF in SR compared to patients with no history of AF (Table 1A). There were nine proteins that differed between AF rhythm and sinus rhythm, of which five (SYNPO2L, MYPN, NACA, PKP2, and CALU) were increased (Table 1B) and four (TUBA8, MYOZ1, CASQ2, and CAMK2D) were decreased. In patients diagnosed with persistent AF in AF rhythm versus those with a history of persistent AF but in SR at surgery, four were increased (SYNPO2L, MYPN, NACA, and PKP2) and two (CASQ2 and CAMK2D) were decreased (Table 1C). Expression of proteins encoded by putative AF risk genes was not different in long-standing persistent AF rhythm compared to persistent AF rhythm. Conclusions: In one of the largest proteomic datasets in human LAA, we found that key proteins encoded by genes associated with AF risk are altered in patients in AF rhythm. Expression of these proteins also differs by AF state. These studies provide insight into pathways that may be targeted for AF prevention and highlight the importance of early intervention to prevent AF progression.

Toll-like receptors (TLRs) are transmembrane proteins that recognize microbial components or cellular danger signals and activate intracellular signaling pathways, leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein that binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed that TLR9 was phosphorylated sequentially on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling.IMPORTANCEToll-like receptors (TLRs) are critical components of cellular innate immune responses to microbial infection or tissue damage. TLRs are transmembrane proteins that require activation to mount a successful host response; TLR mutations are associated with human diseases. TLR ligands are also used as vaccine adjuvants to amplify the inflammatory responses of the host, activation of TLRs, and their regulation are essential. Here, we report the molecular mechanisms of TLR9 activation by tyrosine phosphorylation of its cytoplasmic domain. TLR9 interacts with EGFR and Syk, the tyrosine kinases, which phosphorylate two specific tyrosine residues on the TLR9 cytoplasmic domain. Mutation of these tyrosine residues or deficiency of these tyrosine kinases leads to impaired TLR9 signaling. Therefore, our results elucidate the early events of TLR9 signaling with implications in inflammatory diseases.

Six-transmembrane protein of prostate 4 (Steap4), highly expressed in adipose tissue, is associated with metabolic homeostasis. Dysregulated adipose and mitochondrial metabolism contributes to obesity, highlighting the need to understand their interplay. Whether and how Steap4 influences mitochondrial function, adipocytes, and energy expenditure remain unclear. Adipocyte-specific Steap4-deficient mice exhibited increased fat mass and severe insulin resistance in our high-fat diet model. Mass spectrometry identified two classes of Steap4 interactomes: mitochondrial proteins and proteins involved in splicing. RNA sequencing (RNA-seq) analysis of white adipose tissue demonstrated that Steap4 deficiency altered RNA splicing patterns with enriched mitochondrial functions. Indeed, Steap4 deficiency impaired respiratory chain complex activity, causing mitochondrial dysfunction in white adipose tissue. Consistently, brown adipocyte-specific Steap4 deficiency impaired mitochondrial function, increased brown fat whitening, reduced energy expenditure, and exacerbated insulin resistance in a high-fat model. Overall, our study highlights Steap4's critical role in modulating adipocyte mitochondrial function, thereby controlling thermogenesis, energy expenditure, and adiposity.

In eukaryotes, the last steps of heme biosynthesis occur in mitochondria and so heme must be transported to reach many heme-dependent proteins that mature and function outside this organelle. Although the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has emerged as a key intracellular heme chaperone, how it performs heme deliveries to its numerous clients is poorly understood. It is unknown if handoffs of the GAPDH-bound heme require that it make direct contact with its clients or instead involve GAPDH passing its heme to middlemen proteins to execute the final heme transfers. To address this question, we studied GAPDH heme transfer to the client protein indoleamine 2,3-dioxygenase 1 (IDO1), whose enzyme activity is heme-dependent and regulates mammalian immune responses and cancer progression. A chemical crosslinking-mass spectrometry approach identified two Lys residues that formed an inter-protein crosslink across a previously uncharacterized GAPDH-IDO1 interface. This guided our building a model of the GAPDH-IDO1 complex so we could interrogate by point mutagenesis the role of the GAPDH-IDO1 contact in enabling delivery of GAPDH heme to IDO1. We characterized behaviors of the GAPDH and IDO1 variants in their purified form and when they were expressed in the HEK293T human cell line. This revealed GAPDH heme transfer to IDO1 in cells requires that they make a direct contact which relies on a specific Lys-Asp charge pairing interaction forming across the complex interface. These findings illuminate a key step in the maturation of functional IDO1 and improve our understanding of how GAPDH may perform its heme trafficking function in mammals.

Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, the Schizosaccharomyces pombe ortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling in S. pombe. Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.

Abstract Introduction Given the prevalence and staggering cost of neurological disorders, there is dire need for effective early detection and intervention tools. Emerging evidence suggests that multidisciplinary lifestyle interventions (MLI) may mitigate the risk and progression of neurological disorders. The objectives of this protocol are (1) to test the impact of MLI on the progression of neurological disorders and (2) to identify multi-omic biomarkers for early stages of neurological disease and the impact of MLIs on these biomarkers. Methods and analysis We present the Multidisciplinary lifestyle Interventions for Neurological Disorders during the Silent phase (MINDS) protocol, a randomized controlled trial of MLI in neurologically healthy older adults (≥ 50 years old) exhibiting elevated risk for common neurological disorders: stroke, epilepsy, Parkinson’s Disease, or Alzheimer’s disease and related dementias. Participants will be randomly assigned to intervention (n = 100) or control (n = 100) groups. The intervention group will receive 3 months of weekly 2-hour sessions on diet education, yoga, music therapy, and cognitive skills training. The participants’ neurological health and engagement in relevant lifestyle practices will be assessed at regular intervals for 12 months. Neuroimaging and samples for multi-omic analyses will be collected at baseline, and at 3 months and 12 months after enrollment. Primary outcomes will be signs of progression of the neurological disorder risk that qualified them for study enrollment or a clinical diagnosis of the disorder. Secondary and exploratory outcomes will be based on self-reported health and multi-omic data. Data analysis will include between-group and longitudinal within-group analyses. Perspectives The MINDS protocol and trial aims to clarify the impact of MLI on the progression of neurological disorder risk or diagnosis in older adults and to identify biomarkers that can be used to confirm MLI efficacy. The ability to validate the impact of MLI on neurological disorder progression based on biomarker data allows the identification of individuals most likely to benefit from such therapies in the early stages of neurological disease. Trial registration The trial is registered on the National Institutes of Health (NIH) ClinicalTrials.gov (NCT05984056) site. It was registered on August 2nd, 2023. The trial has full approval of the Cleveland Clinic Internal Review Board.

The induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria-localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia-reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.

ABSTRACT Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatory P. aeruginosa culture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1α, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3' UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3' UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3' UTR reveals a more complex regulation of Sec incorporation than previously realized.