Proteomics Center

Stony Brook, NY, United States

Proteomics Center

Stony Brook, NY, United States
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von Roemeling C.A.,Mayo Clinic Comprehensive Cancer Center | Marlow L.A.,Mayo Clinic Comprehensive Cancer Center | Radisky D.C.,Mayo Clinic Comprehensive Cancer Center | Rohl A.,Mayo Clinic Comprehensive Cancer Center | And 7 more authors.
Oncotarget | Year: 2014

Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 'hits' that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC.


Escobar-Hoyos L.F.,Research Group Genetic Toxicology and Cytogenetics | Koller A.,Proteomics Center | Chen E.I.,Proteomics Center
Modern Pathology | Year: 2014

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio=14.76, P=0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.


Lu X.,Shenzhen University | Lu X.,Proteomics Center | Meima M.E.,Proteomics Center | Dekkers D.H.W.,Proteomics Center | And 3 more authors.
Circulation Research | Year: 2016

Rationale: The (pro)renin receptor ([P]RR) interacts with (pro)renin at concentrations that are >1000× higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin system-independent functions for (P)RR related to its association with the vacuolar H+-ATPase. Objective: To uncover renin-angiotensin system-independent functions of the (P)RR. Methods and Results: We used a proteomics-based approach to purify and identify (P)RR-interacting proteins. This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by coimmunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (P)RR expression in hepatocytes decreased SORT1 and low-density lipoprotein (LDL) receptor protein abundance and, as a consequence, resulted in severely attenuated cellular LDL uptake. In contrast to LDL, endocytosis of epidermal growth factor or transferrin remained unaffected by silencing of the (P)RR. Importantly, reduction of LDL receptor and SORT1 protein abundance occurred in the absence of changes in their corresponding transcript level. Consistent with a post-transcriptional event, degradation of the LDL receptor induced by (P)RR silencing could be reversed by lysosomotropic agents, such as bafilomycin A1. Conclusions: Our study identifies a renin-angiotensin system-independent function for the (P)RR in the regulation of LDL metabolism by controlling the levels of SORT1 and LDL receptor. © 2015 American Heart Association, Inc.


PubMed | University of Cauca, State University of New York at Stony Brook, United States 3 Proteomics Center and Proteomics Center
Type: Journal Article | Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc | Year: 2014

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio=14.76, P=0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.


PubMed | Mayo Clinic Comprehensive Cancer Center, Incogen Inc. and Proteomics Center
Type: Journal Article | Journal: Oncotarget | Year: 2014

Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 hits that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC.


News Article | December 22, 2016
Site: www.eurekalert.org

Making muscles burn more fat and less glucose can increase exercise endurance, but could simultaneously cause diabetes, says a team of scientists from Baylor College of Medicine and other institutions. Mouse muscles use glucose (carbohydrate) as fuel when the animals are awake and active and switch to fat (lipid) when they are asleep. The team discovered that disrupting this natural cycle may lead to diabetes but, surprisingly, also can enhance exercise endurance. The switch is controlled by a molecule called histone deacetylase 3, or HDAC3. This finding opens the possibility of selecting the right time to exercise for losing body fat but also raises the concern of using HDAC inhibitors as doping drugs for endurance exercise. The study appears in Nature Medicine. "How the muscle uses glucose is regulated by its internal circadian clock that anticipates the level of its activity during the day and at night," said senior author Dr. Zheng Sun, assistant professor of medicine - diabetes, endocrinology and metabolism, and of molecular and cellular biology at Baylor. "The circadian clock works by turning certain genes on and off as the 24-hour cycle progresses. HDAC3 is a key connection between the circadian clock and gene expression. Our previous work showed that HDAC3 helps the liver alternate between producing glucose and producing lipid. In this work, we studied how HDAC3 controls the use of different fuels in skeletal muscle." Skeletal muscles, the voluntary muscles, are important in the control of blood glucose in the body. They consume most of the glucose, and if they develop insulin resistance and consequently are not able to use glucose, then diabetes likely will develop. To study the role of HDAC3 in mouse skeletal muscle, Sun and colleagues genetically engineered laboratory mice to deplete HDAC3 only in the skeletal muscles. Then they compared these knocked out mice with normal mice regarding how their muscles burn fuel. When normal mice eat, their blood sugar increases and insulin is released, which stimulates muscles to take in and use glucose as fuel. "When the knocked out mice ate, their blood sugar increased and insulin was released just fine, but their muscles refused to take in and use glucose," said Sun. "Lacking HDAC3 made the mice insulin resistant and more prone to develop diabetes." Yet, when the HDAC3-knocked out mice ran on a treadmill, they showed superior endurance, "which was intriguing because diabetes is usually associated with poor muscle performance," said Sun. "Glucose is the main fuel of muscle, so if a condition limits the use of glucose, the expectation is low performance in endurance exercises. That's the surprise." The researchers then studied what fueled the HDAC3-knocked out mice's stellar performance using metabolomics approaches and found that their muscles break down more amino acids. This changed the muscles' preference from glucose to lipids and allowed them to burn lipid very efficiently. This explains the high endurance, because the body carries a much larger energy reservoir in the form of lipid than carbohydrate. The finding challenges the widely-used carbohydrate-loading (carbo-loading) strategy for improving endurance performance. "Carbo-loading didn't make evolutionary sense before the invention of agriculture," said Sun. "Switching muscles from using carbohydrates to lipids could increase exercise endurance, especially for low-intensity exercise." The study suggests that HDAC inhibitors, a class of small molecule drugs currently being tested for treating several diseases, could potentially be used to manipulate such fuel switch in muscle and therefore raises concern of doping. The team performed a number of functional genomics studies that established the link between HDAC3 and the circadian clock. "In normal mice, when the mouse is awake, the clock in the muscle anticipates a feeding cycle and uses HDAC3 to turn off many metabolic genes. This leads the muscles to use more carbohydrate," said Sun. "When the animal is about to go to sleep and anticipates a fasting cycle, the clock removes HDAC3. This leads the muscles to use more lipid." Although these studies were done in mice, the researchers speculate that human muscles most likely will follow the same cycle. The study opens the possibility of promoting body fat burning by increasing exercise activity during the periods in which muscles use lipid, which is at night for people. "Losing body fat would be easier by exercising lightly and fasting at night," said Sun. "It's not a bad idea to take a walk after dinner." Other contributors to this work include Sungguan Hong, Wenjun Zhou, Bin Fang, Wenyun Lu, Emanuele Loro, Manashree Damle, Guolian Ding, Jennifer Jager, Sisi Zhang, Yuxiang Zhang, Dan Feng, Qingwei Chu, Brian D Dill, Henrik Molina, Tejvir S Khurana, Joshua D Rabinowitz and Mitchell A Lazar. The authors are affiliated with one or more of the following institutions: Baylor College of Medicine, University of Pennsylvania, Princeton University, Shanghai Jiao Tong University and Rockefeller University. Financial support was provided by the National Institutes of Health grants DK043806 and DK099443. The study was also supported by multiple core facilities including the Penn Diabetes Center (DK19525) Functional Genomics Core and Mouse Metabolic Phenotyping Core, Rockefeller University Proteomics Center, Princeton/Penn Regional Metabolomics Core, Vanderbilt MMPC (DK59637) and the Baylor Diabetes Center (DK079638) Mouse Metabolism Core.


Kubben N.,Maastricht University | Kubben N.,U.S. National Institutes of Health | Voncken J.W.,Maastricht University | Demmers J.,Proteomics Center | And 5 more authors.
Nucleus | Year: 2010

The nuclear lamina is an interconnected meshwork of intermediate filament proteins underlying the nuclear envelope. The lamina is an important regulator of nuclear structural integrity as well as nuclear processes, including transcription, DNA replication and chromatin remodeling. The major components of the lamina are A- and B-type lamins. Mutations in lamins impair lamina functions and cause a set of highly tissue-specific diseases collectively referred to as laminopathies. The phenotypic diversity amongst laminopathies is hypothesized to be caused by mutations affecting specific protein interactions, possibly in a tissue-specific manner. Current technologies to identify interaction partners of lamin A and its mutants are hampered by the insoluble nature of lamina components. To overcome the limitations of current technologies, we developed and applied a novel, unbiased approach to identify lamin A-interacting proteins. This approach involves expression of the high-affinity OneSTrEP-tag, precipitation of lamin-protein complexes after reversible protein cross-linking and subsequent protein identification by mass spectrometry. We used this approach to identify in mouse embryonic fibroblasts and cardiac myocyte NklTAg cell lines proteins that interact with lamin A and its mutant isoform progerin, which causes the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HG PS). We identified a total of 313 lamina-interacting proteins, including several novel lamin A interactors, and we characterize a set of 35 proteins which preferentially interact with lamin A or progerin. © 2010 Landes Bioscience.


Van De Nobelen S.,Erasmus MC | Van De Nobelen S.,Max Planck Institute of Immunobiology and Epigenetics | Rosa-Garrido M.,University of Cantabria | Leers J.,Justus Liebig University | And 13 more authors.
Epigenetics and Chromatin | Year: 2010

Background: CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners. Results. Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter. Conclusions. UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription. © 2010 van de Nobelen et al; licensee BioMed Central Ltd.


Roma-Mateo C.,Institute Biomedicina Of Valencia | Solaz-Fuster M.D.C.,Institute Biomedicina Of Valencia | Gimeno-Alcaniz J.V.,Institute Biomedicina Of Valencia | Dukhande V.V.,University of Kentucky | And 8 more authors.
Biochemical Journal | Year: 2011

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser 25 as the residue involved in this modification. We also show that Ser 25 is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser 25 by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser 25 is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients. © The Authors Journal compilation © 2011 Biochemical Society.


Engelen E.,Erasmus MC Wytemaweg | Brandsma J.H.,Erasmus MC Wytemaweg | Moen M.J.,Erasmus MC Wytemaweg | Signorile L.,Erasmus MC Wytemaweg | And 8 more authors.
Nature Communications | Year: 2015

The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. © 2015 Macmillan Publishers Limited. All rights reserved.

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