Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms ...underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and are presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.
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•CNS-derived antigens accumulate at dural sinuses and are captured by dural APCs•Dural sinus-associated APCs present CSF-borne antigens to patrolling T cells•The dural sinus stroma orchestrates T cell trafficking•Immune hubs along dural sinuses allow CNS immunosurveillance
Rustenhoven et al. identify the dural sinuses as a neuroimmune interface, where patrolling T cells survey brain- and CSF-derived antigens to enable CNS immune surveillance. This niche is altered during aging and neuroinflammation and may represent a new therapeutic target for neurological diseases.
Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in
cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that ...MFN2 activity can be determined by Met
and His
interactions with Asp
and Leu
and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser
Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg
→Gln
and MFN2 Thr
→Met
, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr
→Met
mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.
Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and ...relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.
Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ∼30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 ...within the catalytic domain (most commonly R882H), suggesting the possibility of dominant-negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ∼80% compared with the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity, but coexpression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes.
•AML cases with DNMT3A mutations at R882 exhibit focal hypomethylation•R882H DNMT3A is a dominant-negative inhibitor of WT DNMT3A•WT DNMT3A forms stable, active homotetramers•R882H DNMT3A dominantly disrupts DNMT3A tetramerization
Heterozygous DNMT3A R882H mutation is common in acute myeloid leukemia (AML). Russler-Germain et al. show that DNMT3AR882H inhibits wild-type DNMT3A activity in cells, but not in vitro, and that AML cells with the R882H mutation have reduced de novo methyltransferase activity and focal CpG hypomethylation.
Extensive genomic characterization of human cancers presents the problem of inference from genomic abnormalities to cancer phenotypes. To address this problem, we analysed proteomes of colon and ...rectal tumours characterized previously by The Cancer Genome Atlas (TCGA) and perform integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. Messenger RNA transcript abundance did not reliably predict protein abundance differences between tumours. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA 'microsatellite instability/CpG island methylation phenotype' transcriptomic subtype, but had distinct mutation, methylation and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates, including HNF4A (hepatocyte nuclear factor 4, alpha), TOMM34 (translocase of outer mitochondrial membrane 34) and SRC (SRC proto-oncogene, non-receptor tyrosine kinase). Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background:
Anterior cruciate ligament (ACL) tears often result in knee effusion and an increased risk for developing knee osteoarthritis (OA) in the long run. The molecular profile of these ...effusions could be informative regarding initial steps in the development of posttraumatic OA after an ACL tear.
Hypothesis:
The proteomics of knee synovial fluid changes over time after ACL injury.
Study Design:
Descriptive laboratory study.
Methods:
Synovial fluid was collected from patients with an acute traumatic ACL tear presenting to the office for evaluation (18.31 ± 19.07 days from injury) (aspiration 1) and again at the time of surgery (35.41 ± 58.15 days after aspiration 1 (aspiration 2). High-resolution liquid chromatography mass spectrometry was used to assess the quantitative protein profile of synovial fluid, and differences in protein profile between the 2 aspirations were determined computationally.
Results:
A total of 58 synovial fluid samples collected from 29 patients (12 male, 17 female; 12 isolated ACL tear, 17 combined ACL and meniscal tear) with a mean age and body mass index of 27.01 ± 12.78 years and 26.30 ± 4.93, respectively, underwent unbiased proteomics analysis. The levels of 130 proteins in the synovial fluid changed over time (87 high, 43 low). Proteins of interest that were significantly higher in aspiration 2 included CRIP1, S100A11, PLS3, POSTN, and VIM, which represent catabolic/inflammatory activities in the joint. Proteins with a known role in chondroprotection and joint homeostasis such as CHI3L2 (YKL-39), TNFAIP6/TSG6, DEFA1, SPP1, and CILP were lower in aspiration 2.
Conclusion:
Synovial fluid from knees with ACL tears exhibits an increased burden of inflammatory (catabolic) proteins relevant to OA with reduced levels of chondroprotective (anabolic) proteins.
Clinical Relevance:
This study identified a set of novel proteins that provide new biological insights into the aftermath of ACL tears. Elevated inflammation and decreased chondroprotection could represent initial disruption of homeostasis, potentially initiating the development of OA. Longitudinal follow-up and mechanistic studies are necessary to assess the functional role of these proteins in the joint. Ultimately, these investigations could lead to better approaches to predict and possibly improve patient outcomes.
Background:
Anterior cruciate ligament (ACL) tears are associated with posttraumatic osteoarthritis, but the early biological changes that initiate joint degeneration after this injury are not well ...characterized. ACL tears typically result in effusion in the knee, which may provide insight into the initial response of the joint to injuries.
Hypothesis:
Patient- and injury-specific factors are associated with the proteomics of synovial fluid in knees with ACL tears.
Study Design:
Descriptive laboratory study.
Methods:
Synovial fluid was collected from 105 patients (38 male, 67 female) with an acute traumatic ACL tear. Patient- and injury-specific factors such as age, sex, body mass index, time from injury, presence/absence of concomitant meniscal tears, and location of concomitant bone bruises (if present) were recorded. The protein concentration of synovial fluid was measured, followed by benchmarking of samples for multi-affinity high-abundance protein depletion. An isotropically labeled high-resolution nano-liquid chromatography with tandem mass spectrometry–based proteomic approach was used to determine the synovial fluid protein profile. Data were processed, quality controlled, and analyzed computationally for each patient and injury factor.
Results:
The proteomics of synovial fluid from ACL tears was associated with patient sex, injury pattern, and location of bone bruises but not with patient age, body mass index, or time from injury. Knees with an isolated ACL tear had higher glutathione peroxidase 1 (GPX1) and plastin 3 levels than knees with an ACL tear and meniscal tear. A bone bruise on the lateral femoral condyle was associated with elevated leptin and glucose-6-phosphate dehydrogenase (G6PD) levels. A bone bruise on the lateral tibial plateau was associated with decreased GPX1 levels. Male patients had higher matrix metalloproteinase 9 and lower G6PD levels than female patients.
Conclusion:
Patient sex, injury pattern, and bone bruise location were important determinants of the proteomic profile of effusion resulting from ACL tears.
Clinical Relevance:
Longitudinal follow-ups to see if and how proteomic differences relate to clinical outcomes and mechanistic studies to assess the role that specific proteins play in the joint are warranted. Ultimately, these investigations could lead to better approaches to predict clinical outcomes and identify possible interventions to optimize outcomes in these patients.
Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent ...advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies.
Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms ...of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib
A complex in/del frameshift in
decreased sensitivity to buparlisib via NEK9/MAP2K4-dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.
Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition.
.
Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now ...being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.
•Decitabine treatment of in vitro expanded primary AML samples leads to global hypomethylation.•Highly methylated CpGs are most affected by decitabine-induced hypomethylation, with little influence on transcriptional activity.
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