Mitochondrial reprogramming by activating OXPHOS via glutamine metabolism in African American patients with bladder cancer.

Bladder cancer (BLCA) mortality is higher in African American (AA) patients compared with European American (EA) patients, but the molecular mechanism underlying race-specific differences are unknown. To address this gap, we conducted comprehensive RNA-Seq, proteomics, and metabolomics analysis of BLCA tumors from AA and EA. Our findings reveal a distinct metabolic phenotype in AA BLCA characterized by elevated mitochondrial oxidative phosphorylation (OXPHOS), particularly through the activation of complex I. The results provide insight into the complex I activation-driven higher OXPHOS activity resulting in glutamine-mediated metabolic rewiring and increased disease progression, which was also confirmed by [U]13C-glutamine tracing. Mechanistic studies further demonstrate that knockdown of NDUFB8, one of the components of complex I in AA BLCA cells, resulted in reduced basal respiration, ATP production, GLS1 expression, and proliferation. Moreover, preclinical studies demonstrate the therapeutic potential of targeting complex I, as evidenced by decreased tumor growth in NDUFB8-depleted AA BLCA tumors. Additionally, genetic and pharmacological inhibition of GLS1 attenuated mitochondrial respiration rates and tumor growth potential in AA BLCA. Taken together, these findings provide insight into BLCA disparity for targeting GLS1-Complex I for future therapy.

Quantitative proteomics reveals serum proteome alterations during metastatic disease progression in breast cancer patients.

BACKGROUND: Tumor recurrence and metastatic progression remains the leading cause for breast cancer related mortalities. However, the proteomes of patient- matched primary breast cancer (BC) and metastatic lesions have not yet been identified, due to the lack of clinically annotated longitudinal samples. In this study, we evaluated the global-proteomic landscape of BC patients with and without distant metastasis as well as compared the proteome of distant metastatic disease with its corresponding primary BC, within the same patient. METHODS: We performed mass spectrometry-based proteome profiling of 73 serum samples from 51 BC patients. Among the 51 patients with BC, 29 remained metastasis-free (henceforth called non-progressors), and 22 developed metastases (henceforth called progressors). For the 22 progressors, we obtained two samples: one collected within a year of diagnosis, and the other collected within a year before the diagnosis of metastatic disease. MS data were analyzed using intensity-based absolute quantification and normalized before differential expression analysis. Significantly differentially expressed proteins (DEPs; absolute fold-change ≥ 1.5, P-value < 0.05 and 30% abundance per clinical group) were subjected to pathway analyses. RESULTS: We identified 967 proteins among 73 serum samples from patients with BC. Among these, 39 proteins were altered in serum samples at diagnosis, between progressors and non-progressors. Among these, 4 proteins were further altered when the progressors developed distant metastasis. In addition, within progressors, 20 proteins were altered in serum collected at diagnosis versus at the onset of metastasis. Pathway analysis showed that these proteins encoded pathways that describe metastasis, including epithelial-mesenchymal transition and focal adhesion that are hallmarks of metastatic cascade. CONCLUSIONS: Our results highlight the importance of examining matched samples from distant metastasis with primary BC samples collected at diagnosis to unravel subset of proteins that could be involved in BC progression in serum. This study sets the foundation for additional future investigations that could position these proteins as non-invasive markers for clinically monitoring breast cancer progression in patients.

Galectin 3-binding protein (LGALS3BP) depletion attenuates hepatic fibrosis by reducing transforming growth factor-ß1 (TGF-ß1) availability and inhibits hepatocarcinogenesis.

BACKGROUND: Increased Galectin 3-binding protein (LGALS3BP) serum levels have been used to assess hepatic fibrosis stages and the severity of hepatocellular carcinoma (HCC). Considering the crucial role of transforming growth factor-ß1 (TGF-ß1) in the emergence of these diseases, the present study tested the hypothesis that LGALS3BP regulates the TGF-ß1 signaling pathway. METHODS: The expression levels of LGALS3BP and TGFB1 were analyzed in patients with metabolic dysfunction-associated steatohepatitis (MASH) and HCC. Multiple omics techniques, such as RNA-sequencing, transposase-accessible chromatin-sequencing assay, and liquid chromatography-tandem mass spectrometry proteomics, were used to identify the regulatory mechanisms for the LGALS3BP-TGF-ß1 axis. The effects of altered TGF-ß1 signaling by LGALS3BP were investigated in conditional LGALS3BP-knockin and LGALS3BP-knockout mice. RESULTS: In patients with MASH and HCC, the levels of LGALS3BP and TGFB1 exhibited positive correlations. Stimulation of LGALS3BP by the inflammatory cytokine interferon α in HCC cells or ectopic overexpression of LGALS3BP in hepatocytes promoted the expression levels of TGFB1. Aggravated fibrosis was observed in the livers of hepatocyte-specific LGALS3BP-knockin mice, with increased TGFB1 levels. LGALS3BP directly bound to and assembled integrin αV, an integral mediator required for releasing active TGF-ß1 from extracellular latent complex with the rearranged F-actin cytoskeleton. The released TGF-ß1 activated JunB transcription factor, which in turn promoted the TGF-ß1 positive feedback loop. LGALS3BP deletion in the hepatocytes downregulated TGF-ß1 signaling and CCl4 induced fibrosis. Moreover, LGALS3BP depletion hindered hepatocarcinogenesis by limiting the availability of fibrogenic TGF-ß1. CONCLUSION: LGALS3BP plays a crucial role in hepatic fibrosis and carcinogenesis by controlling the TGF-ß1 signaling pathway, making it a promising therapeutic target in TGF-ß1-related diseases.

Sphingosine kinase 2 regulates protein ubiquitination networks in neurons.

Two sphingosine kinase isoforms, sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2), synthesize the lipid sphingosine-1-phosphate (S1P) by phosphorylating sphingosine. SPHK1 is a cytoplasmic kinase, and SPHK2 is localized to the nucleus and other organelles. In the cytoplasm, the SPHK1/S1P pathway modulates autophagy and protein ubiquitination, among other processes. In the nucleus, the SPHK2/S1P pathway regulates transcription. Here, we hypothesized that the SPHK2/S1P pathway governs protein ubiquitination in neurons. We found that ectopic expression of SPHK2 increases ubiquitinated substrate levels in cultured neurons and pharmacologically inhibiting SPHK2 decreases protein ubiquitination. With mass spectrometry, we discovered that inhibiting SPHK2 affects lipid and synaptic protein networks as well as a ubiquitin-dependent protein network. Several ubiquitin-conjugating and hydrolyzing proteins, such as the E3 ubiquitin-protein ligases HUWE1 and TRIP12, the E2 ubiquitin-conjugating enzyme UBE2Z, and the ubiquitin-specific proteases USP15 and USP30, were downregulated by SPHK2 inhibition. Using RNA sequencing, we found that inhibiting SPHK2 altered lipid and neuron-specific gene networks, among others. Genes that encode the corresponding proteins from the ubiquitin-dependent protein network that we discovered with mass spectrometry were not affected by inhibiting SPHK2, indicating that the SPHK2/S1P pathway regulates ubiquitination at the protein level. We also show that both SPHK2 and HUWE1 were upregulated in the striatum of a mouse model of Huntington's disease, the BACHD mice, indicating that our findings are relevant to neurodegenerative diseases. Our results identify SPHK2/S1P as a novel regulator of protein ubiquitination networks in neurons and provide a new target for developing therapies for neurodegenerative diseases.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests that ion homeostasis is a cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption, which was rescued by pharmacological or genetic inhibition of the CCL2-CCR2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-CCL2 and endothelial CCR2 axis as a mechanism controlling BBB integrity and repair, while providing insights for a therapeutic approach against BBB-related CNS disorders.

PCIF1 is partly cytoplasmic, dynamically localizes to stress granules and binds mRNA coding regions upon oxidative stress.

PCIF1 (Phosphorylated CTD-Interacting Factor 1) is the mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N6,2'-O-dimethyladenosine (m6Am) by methylating adenosines at the first transcribed position of capped mRNAs. While previous studies assumed that PCIF1 was nuclear, cell fractionation and immunofluorescence both show that a population of PCIF1 is localized to the cytoplasm. Further, PCIF1 redistributes to stress granules upon oxidative stress. Immunoprecipitation studies with stressed cells show that PCIF1 also physically interacts with G3BP and other stress granule components. In addition, PCIF1 behaves as a stress granule component as it disassociates from stress granules upon recovery from stress. Overexpressing full-length PCIF1 also inhibits stress granule formation, while knocking out PCIF1 slows stress granule disassembly. Next, our enhanced crosslinking and immunoprecipitation (eCLIP) data show that PCIF1 binds mRNAs in their coding sequences rather than cap-proximal regions. Further PCIF1's association with mRNAs increased upon NaAsO2 stress. In contrast to eCLIP data, ChIP-Seq experiments show that PCIF1 is predominantly associated with transcription start sites rather than gene bodies, indicating that PCIF1's association with mature mRNA is not co-transcriptional. Collectively, our data suggest that PCIF1 has cytoplasmic RNA surveillance role(s) independent of transcription-associated cap-adjacent mRNA modification, particularly during the stress response.

Inferring Bladder Cancer Evolution from Mucosal field Effects by Whole-Organ Spatial Mutational, Proteomic, and Metabolomic Mapping.

Multi-platform mutational, proteomic, and metabolomic spatial mapping was used on the whole-organ scale to identify the molecular evolution of bladder cancer from mucosal field effects. We identified complex proteomic and metabolomic dysregulations in microscopically normal areas of bladder mucosa adjacent to dysplasia and carcinoma in situ. The mutational landscape developed in a background of complex defects of protein homeostasis which included dysregulated nucleocytoplasmic transport, splicesome, ribosome biogenesis, and peroxisome. These changes were combined with altered urothelial differentiation which involved lipid metabolism and protein degradations controlled by PPAR. The complex alterations of proteome were accompanied by dysregulation of gluco-lipid energy-related metabolism. The analysis of mutational landscape identified three types of mutations based on their geographic distribution and variant allele frequencies. The most common were low frequency α mutations restricted to individual mucosal samples. The two other groups of mutations were associated with clonal expansion. The first of this group referred to as ß mutations occurred at low frequencies across the mucosa. The second of this group called γ mutations increased in frequency with disease progression. Modeling of the mutations revealed that carcinogenesis may span nearly 30 years and can be divided into dormant and progressive phases. The α mutations developed gradually in the dormant phase. The progressive phase lasted approximately five years and was signified by the advent of ß mutations, but it was driven by γ mutations which developed during the last 2-3 years of disease progression to invasive cancer. Our study indicates that the understanding of complex alterations involving mucosal microenvironment initiating bladder carcinogenesis can be inferred from the multi-platform whole-organ mapping.

Serum Extracellular Vesicle Protein Profiling for Prediction of Corneal Transplant Rejection.

BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.

The Hepatokine Orosomucoid 2 Mediates Beneficial Metabolic Effects of Bile Acids.

Bile acids (BAs) are pleiotropic regulators of metabolism. Elevated levels of hepatic and circulating BAs improve energy metabolism in peripheral organs, but the precise mechanisms underlying the metabolic benefits and harm still need to be fully understood. In the current study, we identified orosomucoid 2 (ORM2) as a liver-secreted hormone (i.e., hepatokine) induced by BAs and investigated its role in BA-induced metabolic improvements in mouse models of diet-induced obesity. Contrary to our expectation, under a high-fat diet (HFD), our Orm2 knockout (Orm2-KO) exhibited a lean phenotype compared with C57BL/6J control, partly due to the increased energy expenditure. However, when challenged with a HFD supplemented with cholic acid, Orm2-KO eliminated the antiobesity effect of BAs, indicating that ORM2 governs BA-induced metabolic improvements. Moreover, hepatic ORM2 overexpression partially replicated BA effects by enhancing insulin sensitivity. Mechanistically, ORM2 suppressed interferon-γ/STAT1 activities in inguinal white adipose tissue depots, forming the basis for anti-inflammatory effects of BAs and improving glucose homeostasis. In conclusion, our study provides new insights into the molecular mechanisms of BA-induced liver-adipose cross talk through ORM2 induction.

Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity.

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMP-activated protein kinase and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin-positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk, and organism longevity.

Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function.

DIRAS3 is an imprinted tumor suppressor gene encoding a GTPase that has a distinctive N-terminal extension (NTE) not found in other RAS proteins. This NTE and the prenylated C-terminus are required for DIRAS3-mediated inhibition of RAS/MAP signaling and PI3K activity at the plasma membrane. In this study, we applied biochemical, biophysical, and computational methods to characterize the structure and function of the NTE. The NTE peptide recognizes phosphoinositides PI(3,4,5)P3 and PI(4,5)P2 with rapid kinetics and strong affinity. Lipid binding induces NTE structural change from disorder to amphipathic helix. Mass spectrometry identified N-myristoylation of DIRAS3. All-atom molecular dynamic simulations predict DIRAS3 could adhere to the membrane through both termini, suggesting the NTE is involved in targeting and stabilizing DIRAS3 on the membrane by double anchoring. Overall, our results are consistent with DIRAS3's function as a tumor suppressor, whereby the membrane-bound DIRAS3 can effectively target PI3K and KRAS at the membrane.

Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

Daam2 phosphorylation by CK2α negatively regulates Wnt activity during white matter development and injury.

Wnt signaling plays an essential role in developmental and regenerative myelination in the central nervous system. The Wnt signaling pathway is composed of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte (OL) development remains unclear. Here, we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt activity during OL development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of OL development, accelerating early differentiation followed by decelerating maturation and myelination. Application toward white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a unique regulatory node in the Wnt pathway that regulates OL development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration.

Identifying the Reactive Metabolites of Tyrosine Kinase Inhibitor Pexidartinib In Vitro Using LC-MS-Based Metabolomic Approaches.

Pexidartinib (PEX, TURALIO), a selective and potent inhibitor of the macrophage colony-stimulating factor-1 receptor, has been approved for the treatment of tenosynovial giant cell tumor. However, frequent and severe adverse effects have been reported in the clinic, resulting in a boxed warning on PEX for its risk of liver injury. The mechanisms underlying PEX-related hepatotoxicity, particularly metabolism-related toxicity, remain unknown. In the current study, the metabolic activation of PEX was investigated in human/mouse liver microsomes (HLM/MLM) and primary human hepatocytes (PHH) using glutathione (GSH) and methoxyamine (NH2OMe) as trapping reagents. A total of 11 PEX-GSH and 7 PEX-NH2OMe adducts were identified in HLM/MLM using an LC-MS-based metabolomics approach. Additionally, 4 PEX-GSH adducts were detected in the PHH. CYP3A4 and CYP3A5 were identified as the primary enzymes responsible for the formation of these adducts using recombinant human P450s and CYP3A chemical inhibitor ketoconazole. Overall, our studies suggested that PEX metabolism can produce reactive metabolites mediated by CYP3A, and the association of the reactive metabolites with PEX hepatotoxicity needs to be further studied.

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain.

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib.

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests pH homeostasis is a new cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption and reactive gliosis, which were both rescued by pharmacological or genetic inhibition of the NO-CCL2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-NO-CCL2 axis as a pivotal mechanism controlling BBB integrity and repair, while providing insights for a novel therapeutic approach against BBB-related CNS disorders.

The cancer testis antigen TDRD1 regulates prostate cancer proliferation by associating with the snRNP biogenesis machinery.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men. TDRD1, a germ cell-specific gene, is erroneously expressed in more than half of prostate tumors, but its role in prostate cancer development remains elusive. In this study, we identified a PRMT5-TDRD1 signaling axis that regulates the proliferation of prostate cancer cells. PRMT5 is a protein arginine methyltransferase essential for small nuclear ribonucleoprotein (snRNP) biogenesis. Methylation of Sm proteins by PRMT5 is a critical initiation step for assembling snRNPs in the cytoplasm, and the final snRNP assembly takes place in Cajal bodies in the nucleus. By mass spectrum analysis, we found that TDRD1 interacts with multiple subunits of the snRNP biogenesis machinery. In the cytoplasm, TDRD1 interacts with methylated Sm proteins in a PRMT5-dependent manner. In the nucleus, TDRD1 interacts with Coilin, the scaffold protein of Cajal bodies. Ablation of TDRD1 in prostate cancer cells disrupted the integrity of Cajal bodies, affected the snRNP biogenesis, and reduced cell proliferation. Taken together, this study represents the first characterization of TDRD1 functions in prostate cancer development and suggests TDRD1 as a potential therapeutic target for prostate cancer treatment.