Browsing by Subject "Proteomics"
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Item A proteomic signature for dementia with Lewy bodies(Elsevier Inc., 2019-03-15) O'Bryant, Sid E.; Ferman, Tanis J.; Zhang, Fan; Hall, James R.; Pedraza, Otto; Wszolek, Zbigniew K.; Como, Tori; Julovich, David A.; Mattevada, Sravan; Johnson, Leigh A.; Edwards, Melissa; Graff-Radford, Neill R.Introduction: We sought to determine if a proteomic profile approach developed to detect Alzheimer's disease would distinguish patients with Lewy body disease from normal controls, and if it would distinguish dementia with Lewy bodies (DLB) from Parkinson's disease (PD). Methods: Stored plasma samples were obtained from 145 patients (DLB n = 57, PD without dementia n = 32, normal controls n = 56) enrolled from patients seen in the Behavioral Neurology or Movement Disorders clinics at the Mayo Clinic, Florida. Proteomic assays were conducted and analyzed as per our previously published protocols. Results: In the first step, the proteomic profile distinguished the DLB-PD group from controls with a diagnostic accuracy of 0.97, sensitivity of 0.91, and specificity of 0.86. In the second step, the proteomic profile distinguished the DLB from PD groups with a diagnostic accuracy of 0.92, sensitivity of 0.94, and specificity of 0.88. Discussion: These data provide evidence of the potential utility of a multitiered blood-based proteomic screening method for detecting DLB and distinguishing DLB from PD.Item Mercapturic Acid Pathway: A Novel Opportunity for Targeting VHL-Mutant Renal Cell Carcinoma(2011-05-01) Dalasanur Nagaprashantina, Lokesh Prasad Gowda; Sanjay Awasthi; Laszlo ProkaiRenal cell carcinoma Renal cell carcinoma (RCC) is one of the top ten leading causes of cancer deaths in USA. The National Cancer Institute’s statistics reveal doubling of the risk of RCC in past 50 years and recent data indicates that RCC contributes to loss of 195,000 person-years of productivity per calendar year making it an important public health problem [1]. Hence, analysis of the specific signaling pathways and networks in aggressive types of RCC and characterization of the mechanisms of action of novel anti-cancer agents that selectively target the processes of oncogenic transformation and tumor progression in kidney has gained momentum in the translational research in renal oncology. RCC was first described by Konig in the year 1826. The proximal renal tubular origin of many renal tumors was confirmed by Robin and Waldeyer in 1855 and 1867, respectively [2]. The major histological sub-types of renal epithelial tumors are comprised of clear cell RCC (75%), papillary type 1 RCC (5%), papillary type 2 RCC (10%), chromophobe RCC (5%) and oncocytoma (5%) [3]. RCC occurs in both sporadic and hereditary forms. The deletions and loss of function mutations in the tumor suppressor von Hippel-Lindau (VHL) gene which is located on chromosome 3p25.3 leading to “VHL syndrome” (Online Mendelian Inheritance in Man, OMIM, catalogue number: 193300) is a major genetic risk factor for the incidence of clear cell RCC, a major sub-type of RCC [4-6]. The VHL syndrome was first described more than a 2 century ago by Treacher Collins and Eugene von Hippel following the observation of familial inheritance pattern of retinal angiomas [7,8]. Later, Arvind Lindau, a neuropathologist, described the incidence of cerebellar hemangioblastomas [9]. The VHL syndrome is also characterized by an increased predisposition to tumors of adrenal glands, inner ear, spine, and pancreatic cysts and hemangioblastomas [10-15]. Hereditary loss of VHL leads to incidence of multifocal, bilateral and highly vascular tumors in kidneys characterized by an aggressive and metastatic course of progression in younger years of life in contrast to sporadic RCC which generally affects mainly elderly population [16]. The VHL mutations have also been detected in many cases of sporadic RCC which in turn reveals the susceptibility of VHL locus to acquired mutations during life time [17]. The life-style and environmental risks for the incidence of sporadic RCC include cigarette smoking, obesity and asbestos exposure [17-20]. The significance of VHL in tumor formation and progression is due to its designated role in oxygen-sensing machinery of the cell which in turn regulates a plethora of cellular proliferative, metabolic and transcriptional processes. The cellular levels of oxygen in drosophila as well as mammals are sensed by a family of hypoxia inducible factors (HIF), proteins characterized by the presence of basic-helix-loop-helix structure and a PAS (dimerization) domain characteristic of many transcription factors. The human HIFs include HIF1-α, HIF1-β, HIF2-α, HIF2-β, HIF3- α and HIF3-β which participate in oxygen dependent re-programming of cellular transcription [21, 22]. The HIF-α subunits have N-terminal transactivation domain (NTAD) and C-terminal transactivation domain (CTAD) which are specifically regulated in oxygen dependent manner [23]. In hypoxic conditions, the transcriptional co-activators like cAMP-response-elementbinding protein (CREB)-binding protein (CBP) and p300 bind to CTAD of HIF-α. This leads to 3 activation of hypoxia inducible genes like EGFR, VEGF, PDGFβ and TGFα which in turn contribute to enhanced angiogenic and mitogenic potential to survive in hypoxic environment [24-27]. The VHL gene product, pVHL, plays a vital role in regulating the function of HIF-α when oxygen levels are normal in cells. In normoxic conditions, the HIF-α proteins are hydroxylated at asparagine residue in NTAD by factor inhibiting HIF-α (FIH) and at proline residues by HIF prolyl-hydroxylases [28,29]. The prolyl hydroxylation of HIF-α at NTAD leads to binding of pVHL-elongin-cullin2 complex to HIF-α followed by polyubiquitination and proteosomal degradation [29-33]. Thus, pVHL contributes to inhibition of HIF-α signaling. The function of pVHL extends beyond regulation of hypoxic signaling in the cells. The pVHL is required for regulation of integrins and tight junctions in epithelial cells and VHL mutant (VHL-mut) cells have increased levels of HIF2-α, α5-integrin, cyclin D1 and lower p27 levels along with loss of epithelial morphology [34]. The introduction of pVHL into VHL-mut RCC leads to cell cycle arrest, epithelial differentiation and suppression of tumor forming ability [35, 36]. Thus, pVHL plays a vital role in regulating multiple signaling processes of importance in oncogenic transformation, survival of tumors in hypoxic conditions along with a role in maintaining epithelial phenotype.Item Molecular Mechanisms of High-Altitude Acclimatization(MDPI, 2023-01-22) Mallet, Robert T.; Burtscher, Johannes; Pialoux, Vincent; Pasha, Qadar; Ahmad, Yasmin; Millet, Gregoire P.; Burtscher, MartinHigh-altitude illnesses (HAIs) result from acute exposure to high altitude/hypoxia. Numerous molecular mechanisms affect appropriate acclimatization to hypobaric and/or normobaric hypoxia and curtail the development of HAIs. The understanding of these mechanisms is essential to optimize hypoxic acclimatization for efficient prophylaxis and treatment of HAIs. This review aims to link outcomes of molecular mechanisms to either adverse effects of acute high-altitude/hypoxia exposure or the developing tolerance with acclimatization. After summarizing systemic physiological responses to acute high-altitude exposure, the associated acclimatization, and the epidemiology and pathophysiology of various HAIs, the article focuses on molecular adjustments and maladjustments during acute exposure and acclimatization to high altitude/hypoxia. Pivotal modifying mechanisms include molecular responses orchestrated by transcription factors, most notably hypoxia inducible factors, and reciprocal effects on mitochondrial functions and REDOX homeostasis. In addition, discussed are genetic factors and the resultant proteomic profiles determining these hypoxia-modifying mechanisms culminating in successful high-altitude acclimatization. Lastly, the article discusses practical considerations related to the molecular aspects of acclimatization and altitude training strategies.Item Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins(Frontiers Media S.A., 2023-03-28) Ghaly, Maria; Proulx, Jessica; Borgmann, Kathleen; Park, In-WooOur recent data established that HIV-1 Nef is pivotal in determining the fate of cellular proteins by modulating ubiquitination. However, it is unknown which proteins are ubiquitinated in the presence of Nef, a question critical for understanding the proliferation/restriction strategies of HIV-1 in infected cells. To identify cellular proteins ubiquitinated by Nef, we conducted a proteomic analysis of cellular proteins in the presence and absence of Nef. Proteomic analysis in HEK293T cells indicated that 93 proteins were upregulated and 232 were downregulated in their ubiquitination status by Nef. Computational analysis classified these proteins based on molecular function, biological process, subcellular localization, and biological pathway. Of those proteins, we found a majority of molecular functions to be involved in binding and catalytic activity. With respect to biological processes, a significant portion of the proteins identified were related to cellular and metabolic processes. Subcellular localization analysis showed the bulk of proteins to be localized to the cytosol and cytosolic compartments, which is consistent with the known function and location of Nef during HIV-1 infection. As for biological pathways, the wide range of affected proteins was denoted by the multiple modes to fulfill function, as distinguished from a strictly singular means, which was not detected. Among these ubiquitinated proteins, six were found to directly interact with Nef, wherein two were upregulated and four downregulated. We also identified 14 proteins involved in protein stability through directly participating in the Ubiquitin Proteasome System (UPS)-mediated proteasomal degradation pathway. Of those proteins, we found six upregulated and eight downregulated. Taken together, these analyses indicate that HIV-1 Nef is integral to regulating the stability of various cellular proteins via modulating ubiquitination. The molecular mechanisms directing Nef-triggered regulation of cellular protein ubiquitination are currently under investigation.Item Potential two-step proteomic signature for Parkinson's disease: Pilot analysis in the Harvard Biomarkers Study(Elsevier Inc., 2019-05-02) O'Bryant, Sid E.; Edwards, Melissa; Zhang, Fan; Johnson, Leigh A.; Hall, James R.; Kuras, Yuliya; Scherzer, Clemens R.Introduction: We sought to determine if our previously validated proteomic profile for detecting Alzheimer's disease would detect Parkinson's disease (PD) and distinguish PD from other neurodegenerative diseases. Methods: Plasma samples were assayed from 150 patients of the Harvard Biomarkers Study (PD, n = 50; other neurodegenerative diseases, n = 50; healthy controls, n = 50) using electrochemiluminescence and Simoa platforms. Results: The first step proteomic profile distinguished neurodegenerative diseases from controls with a diagnostic accuracy of 0.94. The second step profile distinguished PD cases from other neurodegenerative diseases with a diagnostic accuracy of 0.98. The proteomic profile differed in step 1 versus step 2, suggesting that a multistep proteomic profile algorithm to detecting and distinguishing between neurodegenerative diseases may be optimal. Discussion: These data provide evidence of the potential use of a multitiered blood-based proteomic screening method for detecting individuals with neurodegenerative disease and then distinguishing PD from other neurodegenerative diseases.Item Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma(MDPI, 2023-08-26) Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloElevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.Item QUANTITATIVE PROTEOMIC INVESTIGATION OF ESTROGENIC ENDOCRINE-DISRUPTING EFFECTS IN THE RAT UTERUS USING SYSTEMS BIOLOGY(2014-03) Sahyouni, Fatima; Szarka, Szabolcs; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloEndocrine disrupting chemicals (EDCs) are a class of chemicals that interfere with the biological actions of hormones, and there has been significant public concern about EDCs in the environment adversely affecting both wildlife and human health. They can alter processes regardless of whether they are related to reproduction. The mammalian uterus is one of the most sensitive organs for estrogenicity, but the widely used uterotrophic rat assay to assess known and potential EDCs merely considers the uterine weight gain endpoint. In this presentation, we focus on the quantitative proteomic investigation of estrogenic endocrine disruption in the rat uterus utilizing a comprehensive systems biology approach. Using 17β-estradiol (E2), an endogenous estrogen, will serve as a reference for subsequent studies of agents with estrogenic potential. Purpose (a): To validate potential markers of estrogenicity of discovery identified differentially expressed estrogen-induced proteins in rat uterine tissue using quantitative proteomics. Methods (b): Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap–Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite UniProt rat protein database using the Mascot software. Quantitation was performed using an MS-based total precursor intensity approach using the Scaffold software. Additionally, the differentially expressed proteins were mapped to signaling networks and biological processes using Ingenuity Pathway Analysis (IPA). Results (c): The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2’s uterotrophic effects for our subsequent proteomics study. Estrogen-regulated proteins were identified using an MS-based label-free quantitative approach. With p<0.05 considered statistically significant and >2-fold change as threshold, 135 proteins were differentially regulated by the hormone. Of these significantly differentially regulated proteins, 97 were up-regulated in E2-treated uteri and 38 were down-regulated in E2-treated uteri. When these 135 proteins were submitted for bioinformatic pathway analysis, 131 proteins were mapped into 14 networks that merged into E2-regulated pathways. Major molecular processes involve metabolic pathways, steroid signaling, and inflammatory signaling. Top networks include post-translational modification, protein folding, carbohydrate metabolism, cell death and survival, cancer, and endocrine system disorders. Implicated diseases include endocrine system and metabolic disorders. Proteotypic peptides from proteins that were strongly influenced by E2 administration have been selected for targeted validation studies. Conclusions (d): In addition to confirming the expected increase in wet uterine weights, we have derived interaction networks that mechanistically dissect E2’s uterotrophic effect at the proteome level. We have selected proteotypic peptides of strongly regulated proteins for future targeted validation as a potential biomarker panel for estrogenicity. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535).Item RAPID LABEL-FREE QUANTITATIVE PROTEOMICS OF ESTROGENIC ENDOCRINE-DISRUPTING EFFECTS IN THE RAT UTERUS USING A SYSTEMS BIOLOGY APPROACH(2013-04-12) Sahyouni, FatimaPurpose: To identify estrogen-induced differential protein expression in rat uterine tissue using a rapid label-free proteomics and systems biology approach. Methods: Ovariectomized rats were treated short-term with subcutaneous E2 injections using corn oil as a vehicle. Approximately 10 mg of tissue were dissected from the uterus of vehicle-treated control and E2-treated animals for proteomic analyses. Uterine proteins were extracted with 8M urea for 30 minutes and subsequently processed by reduction, alkylation and digestion for mass spectrometry analysis. The samples were analyzed using a hybrid linear ion trap-Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source and connected to a nanoflow liquid chromatography system. MS/MS data was searched against a composite IPI rat protein database containing both forward and randomized sequences using the Mascot software. Quantitation was performed using an MS/MS-based total ion currents (TICs) approach using the Scaffold software. Additionally, Ingenuity Pathway Analysis (IPA) was utilized to derive interaction networks among the identified proteins. Results: The mammalian uterus increases its weight due to fluid imbibition and cell proliferation by exogenously administered estrogenic compounds. With the observation of weight gain in the treated uterus compared to non-treated control rats, we confirmed E2's uterotrophic effects for our subsequent proteomics study. Out of a total of 262 identified proteins, 163 proteins were differentially regulated (with p<0.05 considered statistically significant) by the hormone. Of the 163 proteins that were significantly regulated, 153 were up-regulated in E2-treated uteri and 10 were down-regulated in E2-treated uteri. These 163 proteins were submitted and mapped into 19 networks that merged into E2-regulated pathways. Top networks included molecular transport, carbohydrate metabolism, cancer, developmental disorders and cellular function and maintenance. Implicated diseases were endocrine system and metabolic disorders. Top signaling pathways involved metabolic pathways, steroid signaling and actin cytoskeleton signaling. Conclusions: In addition to the expected increase in wet uterine weights, we have elucidated and organized a large number of E2-induced protein expression changes into interaction networks. Metabolism and developmental disorders were implicated as the top networks. (Supported by the Robert A. Welch Foundation, BK-0031, and the NIH grant AG031535)