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Biology subjects

Zahedi, R. P.

Publications and source records attributed to Zahedi, R. P..

6 recordsLinked to original sources

CK2 variant function and disease modelling in Drosophila reveal allelic heterogeneity and Wnt/β-catenin-mediated phenotypes

Heterozygous pathogenic variants in CSNK2A1 or CSNK2B encoding the Casein Kinase 2 (CK2) protein complex, lead to pediatric neurodevelopmental disorders, Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS). OCNDS and POBINDS are characterized by a range of symptoms, including developmental delay, intellectual disability, facial dysmorphism, and seizures. Despite over 250 reported cases of OCNDS and POBINDS, we do not fully understand how specific alterations in CK2 relate to the heterogeneity observed in patients. To investigate this, we used the fruit fly, Drosophila melanogaster, as a model system. To assess variant impact, we co-expressed human CSNK2A1 and CSNK2B reference or disease-causing variants in flies. In parallel, we determined the role of Drosophila CkII in the developing and mature nervous system, specifically in neurons and glia. We found that 12/13 variants tested act as full or partial loss-of-function with one CSNK2A1 variant showing gain-of-function. Phospho-proteomic studies in neurons revealed separate signatures for loss- and gain-of-function variants. We found that neuronal and glial CkII is critical for organismal development. Reduction of neuronal CkII in the adult nervous system causes motor and seizure-like phenotypes. Finally, given the known role of CK2 in potentiating Wnt/{beta}-catenin signalling, we show that Wnt agonists partially rescue phenotypes associated with adult-specific neuronal reduction of CkII. This work generates Drosophila models of CSNK2A1 and CSNK2B expression to functionally assess variant impact, as well as an adult-specific neuronal loss-of-function model for drug screening and mechanistic studies.

genetics↗

SysQuan: repurposing SILAC mice for the affordable absolute quantitation of the human proteome

Relative quantitation, used by most MS-based proteomics laboratories to determine protein fold-changes, requires samples being processed and analyzed together for best comparability through minimizing batch differences. This limits the adoption of MS-based proteomics in population-wide studies, and the detection of subtle but relevant changes in heterogeneous samples. Absolute quantitation circumvents these limitations and enables comparison of results across laboratories, studies, and longitudinally. However, high costs of the essential stable isotope labeled (SIL) standards prevents widespread access and limits the number of quantifiable proteins. Our new approach, called "SysQuan", repurposes SILAC mouse tissues/biofluids as system-wide internal standards for matched human samples to enable absolute quantitation of, theoretically, two-thirds of the human proteome using 157,086 shared tryptic peptides. We demonstrate that SysQuan enables quantification of 70% and 31% of the liver and plasma proteomes, respectively. We demonstrate for 14 metabolic proteins that abundant SIL mouse tissues enable cost-effective reverse absolute quantitation in, theoretically, 1000s of human samples. Moreover, 10,000s of light/heavy doublets in untargeted SysQuan datasets enable unique post-acquisition absolute quantitation. SysQuan empowers researchers to replace relative quantitation with affordable absolute quantitation at scale, making data comparable across laboratories, diseases and tissues, enabling completely novel study designs and increasing reusability of data in repositories. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/622109v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c7a58org.highwire.dtl.DTLVardef@1344320org.highwire.dtl.DTLVardef@2321e3org.highwire.dtl.DTLVardef@85bf2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Identification of novel myokines and putative protein targets that mediate functional adaptations in response to chronic contractile activity induced skeletal muscle-extracellular vesicle treatment

We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs. control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.

cell biology↗

Proteomic evidence of astrocytic dysfunction in the olfactory bulb of depressed suicides

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p<0.05) between depressed cases and controls. Gene ontology and gene enrichment analyses suggested that these proteins are involved in biological processes including the complement and coagulation cascades. Cell type enrichment analysis displayed a significant reduction in several canonical astrocytic proteins in OBs from depressed patients. Furthermore, using RNA-fluorescence in-situ hybridization, we observed a decrease in the percentage of ALDH1L1+ cells expressing canonical astrocytic markers including ALDOC, NFIA, GJA1 (connexin 43) and SLC1A3 (EAAT1). These results are consistent with previous reports of downregulated astrocytic marker expression in other brain regions in depressed patients. We also conducted a comparative phosphoproteomic analysis of OB samples and found a dysregulation of proteins involved in neuronal and astrocytic functions. To determine whether OB astrocytic abnormalities is specific to humans, we also performed proteomics on the OB of socially defeated male mice, a commonly used model of depression. Cell-type specific analysis revealed that in socially defeated animals, the most striking OB protein alterations were associated with oligodendrocyte- lineage cells rather than with astrocytes, highlighting an important species difference. Overall, this study further highlights cerebral astrocytic abnormalities as a consistent feature of depression in humans.

neuroscience↗

Clinical proteomics reveals vulnerabilities in non-invasive breast ductal carcinoma and drives personalized treatment strategies.

Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The lack of validated prognostic markers, limited patient numbers and variable tissue quality significantly impact diagnosis, risk stratification, patient enrolment, and results of clinical studies. We performed label-free quantitative proteomics on 50 clinical formalin-fixed, paraffin embedded biopsies, validating 22 putative biomarkers from independent genetic studies. Our comprehensive proteomic phenotyping reveals more than 380 differentially expressed proteins and metabolic vulnerabilities, that can inform new therapeutic strategies for DCIS and IDC. Due to the readily druggable nature of proteins and metabolites, this study is of high interest for clinical research and pharmaceutical industry. To further evaluate our findings, and to promote the clinical translation of our study, we developed a highly multiplexed targeted proteomics assay for 90 proteins associated with cancer metabolism, RNA regulation and signature cancer pathways, such as Pi3K/AKT/mTOR and EGFR/RAS/RAF.

cancer biology↗

Insulin receptors in pancreatic acinar cells contribute to KrasG12D-driven cancer initiation in the context of diet-induced obesity

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly. Here, we demonstrate that insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are dispensable for glucose homeostasis, but necessary for hyperinsulinemia-driven PanIN formation in the context of diet-induced hyperinsulinemia and obesity. Mechanistically, this was attributed to amplified digestive enzyme protein translation, triggering of local inflammation and PanIN metaplasia in vivo. In vitro, insulin dose-dependently increased acinar-to-ductal metaplasia formation in a trypsin- and Insr-dependent manner. Collectively, our data shed light on the mechanisms connecting obesity-driven hyperinsulinemia and pancreatic cancer development.

cancer biology↗