Publications by authors named "Michael A Freitas"

82 Publications

Fibroblast-Specific Proteo-Transcriptomes Reveal Distinct Fibrotic Signatures of Human Sinoatrial Node in Non-Failing and Failing Hearts.

Circulation 2021 Apr 20. Epub 2021 Apr 20.

Department of Physiology & Cell Biology; Bob and Corrine Frick Center for Heart Failure and Arrhythmia, Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH.

Up to fifty percent of the adult human sinoatrial node (SAN), is composed of dense connective tissue. Cardiac diseases including heart failure (HF) may further increase fibrosis within the SAN pacemaker complex, leading to impaired automaticity and conduction of electrical activity to the atria. However, unlike the role of cardiac fibroblasts in pathological fibrotic remodeling and tissue repair, nothing is known about fibroblasts that maintain the inherently fibrotic SAN environment. Intact SAN pacemaker complex was dissected from cardioplegically arrested explanted non-failing (non-HF, n=22; 48.7±3.1y.o,) and HF human hearts (n=16; 54.9±2.6y.o.). Connective tissue content was quantified from Masson's trichrome stained head-center and center-tail SAN sections. Expression of extracellular matrix (ECM) proteins, including Collagens 1, 3A1, cartilage intermediate layer protein 1 (CILP1) and periostin, fibroblast and myofibroblast numbers were quantified by in situ and in vitro immunolabeling. Fibroblasts from the central intramural SAN pacemaker compartment (~10x5x2 mm) and right atria (RA) were isolated, cultured, passaged once, and treated ±transforming growth factor beta-1 (TGFβ1) and subjected to comprehensive high-throughput next-generation sequencing of whole transcriptome, microRNA and proteomic analyses. Intranodal fibrotic content was significantly higher in SAN pacemaker complex from HF vs non-HF hearts (57.7±2.6% vs 44.0±1.2% <0.0001). Proliferating phosphorylated histone3/vimentin/CD31 fibroblasts were higher in HF SAN. Vimentin/alpha smooth muscle actin/CD31 myofibroblasts along with increased interstitial periostin expression were found only in HF SAN. RNA sequencing and proteomic analyses identified unique differences in mRNA, long non-coding RNA, microRNA and proteomic profiles between non-HF and HF SAN and RA fibroblasts, and TGFβ1-induced myofibroblasts. Specifically, proteins and signaling pathways associated with ECM flexibility, stiffness, focal adhesion and metabolism were altered in HF SAN fibroblasts compared to non-HF SAN. This study revealed increased SAN-specific fibrosis with presence of myofibroblasts, CILP1 and periostin-positive interstitial fibrosis only in HF vs non-HF human hearts. Comprehensive proteo-transcriptomic profiles of SAN fibroblasts identified upregulation of genes and proteins promoting stiffer SAN ECM in HF hearts. Fibroblast-specific profiles generated by our proteo-transcriptomic analyses of the human SAN, provide a comprehensive framework for future studies to investigate the role of SAN-specific fibrosis in cardiac rhythm regulation and arrhythmias.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051583DOI Listing
April 2021

Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.

Pancreas 2021 01;50(1):17-28

From the Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine.

Objectives: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influence the integrity and quality of proteins and nucleic acids.

Methods: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection; after 1, 2, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas nucleic acid integrity values determined DNA and RNA degradation.

Results: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples.

Conclusions: Adding PIs immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.
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http://dx.doi.org/10.1097/MPA.0000000000001717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883383PMC
January 2021

Fractionation of DNA and protein from individual latent fingerprints for forensic analysis.

Forensic Sci Int Genet 2021 Jan 21;50:102405. Epub 2020 Oct 21.

Signature Science, LLC, Austin, TX, USA.

Human touch samples represent a significant portion of forensic DNA casework. Yet, the generally low abundance of genetic material combined with the predominantly extracellular nature of DNA in these samples makes DNA-based forensic analysis exceptionally challenging. Human proteins present in these same touch samples offer an abundant and environmentally-robust alternative. Proteogenomic methods, using protein sequence variants arising from nonsynonymous DNA mutations, have recently been applied to forensic analysis and may represent a viable option looking forward. However, DNA analysis remains the gold standard and any proteomics-based methods would need to consider how DNA could be co-extracted from samples without significant loss. Herein, we describe a simple workflow for the collection, enrichment and fractionation of DNA and protein in latent fingerprint samples. This approach ensures that DNA collected from a latent fingerprint can be analyzed by traditional DNA casework methods, while protein can be proteolytically digested and analyzed via standard liquid chromatography-tandem mass spectrometry-based proteomics methods from the same touch sample. Sample collection from non-porous surfaces (i.e., glass) is performed through the application of an anionic surfactant over the fingermark. The sample is then split into separate DNA and protein fractions following centrifugation to enrich the protein fraction by pelleting skin cells. The results indicate that this workflow permits analysis of DNA within the sample, yet highlights the challenge posed by the trace nature of DNA in touch samples and the potential for DNA to degrade over time. Protein deposited in touch samples does not appear to share this limitation, with robust protein quantities collected across multiple human donors. The quantity and quality of protein remains robust regardless of fingerprint age. The proteomic content of these samples is consistent across individual donors and fingerprint age, supporting the future application of genetically variable peptide (GVP) analysis of touch samples for forensic identification.
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http://dx.doi.org/10.1016/j.fsigen.2020.102405DOI Listing
January 2021

Proteomic characterization of a trauma-based rat model of heterotopic ossification identifies interactive signaling networks as potential therapeutic targets.

J Proteomics 2020 08 21;226:103907. Epub 2020 Jul 21.

Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, College of Medicine and Arthur G. James Comprehensive Cancer Center, Columbus, OH, United States of America. Electronic address:

Heterotopic ossification (HO) is the formation of ectopic bone in soft tissues observed in patients following blast injuries, orthopedic or head trauma, burns, or in the context of inborn mutations of genes involved in osteogenesis. There is no universally accepted therapy for HO. This study has used global unbiased mass spectrometry proteomic approaches, validated by western immunoblots, to interrogate skeletal muscle tissues obtained from a highly reproducible rat model of trauma induced HO. During early the phase of HO development, statistically significant modulation of proteins within the following pathways was identified: coagulation, cyclic AMP, extracellular matrix, immunity/inflammation, NADH metabolism, TGFβ. These metabolic proteins and pathways have the potential to serve as diagnostic, prognostic, and therapeutic targets for this devastating orthopedic condition that has considerable impact on the patient's quality of life. Furthermore, the findings confirm and extend previous in vitro stromal/stem cell and clinical studies from the field. SIGNIFICANCE: This study confirms and extends the field's understanding of the protein pathways that are modulated in a rat model of trauma induced heterotopic ossification. The identification of specific proteins such as the AP1 transcription factor as well as protein families such as the complement/coagulation pathway and serine protease inhibitors as biomarkers have potential clinical translational value. These outcomes have relevance to the physiological and pathological mineralization processes contributing to the recovery of orthopedic trauma patients.
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http://dx.doi.org/10.1016/j.jprot.2020.103907DOI Listing
August 2020

Histone acetyltransferase 1 is required for DNA replication fork function and stability.

J Biol Chem 2020 06 4;295(25):8363-8373. Epub 2020 May 4.

Department of Biological Chemistry and Pharmacology, the Ohio State University, Columbus, Ohio, USA

The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
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http://dx.doi.org/10.1074/jbc.RA120.013496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307201PMC
June 2020

Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.

Sci Rep 2020 04 28;10(1):7138. Epub 2020 Apr 28.

Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, USA.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.
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http://dx.doi.org/10.1038/s41598-020-64047-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188826PMC
April 2020

An algorithm for random match probability calculation from peptide sequences.

Forensic Sci Int Genet 2020 07 6;47:102295. Epub 2020 Apr 6.

Center for Human Identification, University of North Texas Health Science Center, Fort Worth, TX, United states.

For the past three decades, forensic genetic investigations have focused on elucidating DNA signatures. While DNA has a number of desirable properties (e.g., presence in most biological materials, an amenable chemistry for analysis and well-developed statistics), DNA also has limitations. DNA may be in low quantity in some tissues, such as hair, and in some tissues it may degrade more readily than its protein counterparts. Recent research efforts have shown the feasibility of performing protein-based human identification in cases in which recovery of DNA is challenged; however, the methods involved in assessing the rarity of a given protein profile have not been addressed adequately. In this paper an algorithm is proposed that describes the computation of a random match probability (RMP) resulting from a genetically variable peptide signature. The approach described herein explicitly models proteomic error and genetic linkage, makes no assumptions as to allelic drop-out, and maps the observed proteomic alleles to their expected protein products from DNA which, in turn, permits standard corrections for population structure and finite database sizes. To assess the feasibility of this approach, RMPs were estimated from peptide profiles of skin samples from 25 individuals of European ancestry. 126 common peptide alleles were used in this approach, yielding a mean RMP of approximately 10.
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http://dx.doi.org/10.1016/j.fsigen.2020.102295DOI Listing
July 2020

The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

Nat Commun 2019 11 25;10(1):5351. Epub 2019 Nov 25.

Center for RNA Medicine, Department of Clinical Medicine, Aalborg University, Copenhagen, Denmark.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.
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http://dx.doi.org/10.1038/s41467-019-13259-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877618PMC
November 2019

A Microfluidic Chip Enables Isolation of Exosomes and Establishment of Their Protein Profiles and Associated Signaling Pathways in Ovarian Cancer.

Cancer Res 2019 07 16;79(13):3503-3513. Epub 2019 May 16.

Division of Gynecologic Oncology, Department of Obstetrics/Gynecology, Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, Ohio.

Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. SIGNIFICANCE: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200082PMC
July 2019

Isolation of Proteins on Nascent Chromatin and Characterization by Quantitative Mass Spectrometry.

Methods Mol Biol 2019 ;1983:17-27

Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, USA.

Replication-coupled chromatin assembly is a very dynamic process that involves not only the replication fork machinery but also chromatin-related factors such as histones, histone chaperones, histone-modifying enzymes, and chromatin remodelers which ensure not only that the genetic information is properly replicated but also that the epigenetic code is reestablished in the daughter cell. Of the histone modifications associated with chromatin assembly, acetylation is the most abundant. Determining how newly synthesized histones get acetylated and what factors affect this modification is vital to understanding how cells manage to properly duplicate the epigenome. Here we describe a combination of the iPOND, quantitative mass spectrometry, and SILAC methodologies to study the protein composition of newly assembled chromatin and the modification state of the associated histones.
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http://dx.doi.org/10.1007/978-1-4939-9434-2_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328701PMC
January 2020

Middle-Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms.

Proteomics 2018 06;18(11):e1700442

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.

Post-translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle-down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) for on-line liquid chromatography-mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to unambiguously distinguish between acetylation and tri-methylation (∆m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono- and di-methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle-dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.
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http://dx.doi.org/10.1002/pmic.201700442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087174PMC
June 2018

The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

J Biol Chem 2018 06 4;293(22):8394-8409. Epub 2018 Apr 4.

From the Departments of Internal Medicine,

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.
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http://dx.doi.org/10.1074/jbc.RA117.001078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986221PMC
June 2018

Author Correction: Nitric oxide mediated inhibition of antigen presentation from DCs to CD4 T cells in cancer and measurement of STAT1 nitration.

Sci Rep 2018 Mar 6;8(1):4203. Epub 2018 Mar 6.

Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, United States.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-21306-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840347PMC
March 2018

Trauma induced heterotopic ossification patient serum alters mitogen activated protein kinase signaling in adipose stem cells.

J Cell Physiol 2018 09 10;233(9):7035-7044. Epub 2018 Apr 10.

Tulane University School of Medicine, Center for Stem Cell Research and Regenerative Medicine, New Orleans, Louisiana.

Post-traumatic heterotopic ossification (HO) is the formation of ectopic bone in non-osseous structures following injury. The precise mechanism for bone development following trauma is unknown; however, early onset of HO may involve the production of pro-osteogenic serum factors. Here we evaluated serum from a cohort of civilian and military patients post trauma to determine early induction gene signatures in orthopaedic trauma induced HO. To test this, human adipose derived stromal/stem cells (hASCs) were stimulated with human serum from patients who developed HO following trauma and evaluated for a gene panel with qPCR. Pathway gene analysis ontology revealed that hASCs stimulated with serum from patients who developed HO had altered gene expression in the activator protein 1 (AP1) and AP1 transcriptional targets pathways. Notably, there was a significant repression in FOS gene expression in hASCs treated with serum from individuals with HO. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway was activated in hASCs following serum exposure from individuals with HO. Serum from both military and civilian patients with trauma induced HO had elevated downstream genes associated with the MAPK pathways. Stimulation of hASCs with known regulators of osteogenesis (BMP2, IL6, Forskolin, and WNT3A) failed to recapitulate the gene signature observed in hASCs following serum stimulation, suggesting non-canonical mechanisms for gene regulation in trauma induced HO. These findings provide new insight for the development of HO and support ongoing work linking the systemic response to injury with wound specific outcomes.
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http://dx.doi.org/10.1002/jcp.26504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083017PMC
September 2018

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4 T cells in cancer and measurement of STAT1 nitration.

Sci Rep 2017 11 13;7(1):15424. Epub 2017 Nov 13.

Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, United States.

Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4 T cells through Jak-STAT signal transduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhibit DC antigen presentation. Antigen presentation from DCs to CD4 T cells (T cell receptor transgenic OT-II) was measured via a [H]-thymidine incorporation proliferation assay. MDSC from melanoma tumor models decreased the levels of proliferation more than pancreatic cancer derived MDSC. T cell proliferation was restored when MDSC were treated with inhibitors of inducible nitric oxide synthase (L-NAME and NCX-4016). A NO donor inhibited OT II T cell receptor recognition of OT II specific tetramers, thus serving as a direct measure of NO inhibition of antigen presentation. Our group has previously demonstrated that STAT1 nitration also mediates MDSC inhibitory effects on immune cells. Therefore, a novel liquid chromatography-tandem mass spectrometry assay demonstrated that nitration of the STAT1-Tyr701 occurs in PBMC derived from both pancreatic cancer and melanoma patients.
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http://dx.doi.org/10.1038/s41598-017-14970-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684213PMC
November 2017

Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly.

Nucleic Acids Res 2017 Sep;45(16):9319-9335

Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210, USA.

Histone acetyltransferase 1 (Hat1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from Hat1+/+ and Hat1-/- mouse embryos, we demonstrate that Hat1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize Hat1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from Hat1-/- cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that Hat1-dependent acetylation of H4 is directly involved in their recruitment. Hat1-/- nascent chromatin is enriched for topoisomerase 2α and 2β. The enrichment of topoisomerase 2 is functionally relevant as Hat1-/- cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that Hat1 is required for proper chromatin topology. In addition, our results indicate that Hat1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.
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http://dx.doi.org/10.1093/nar/gkx545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766187PMC
September 2017

The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer.

J Proteomics 2017 03 27;156:94-103. Epub 2017 Jan 27.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; Division of Medical Oncology, Department of Internal Medicine, College of Medicine; The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Cruciferous vegetable intake is associated with reduced risk of bladder cancer, yet mechanisms remain unclear. Cruciferous vegetable isothiocyanates (ITCs), namely sulforaphane (SFN) and erucin (ECN), significantly inhibit histone deacetylase (HDAC) activity in human bladder cancer cells representing superficial to invasive biology (59-83% inhibition with 20μM, 48h treatment), and in bladder cancer xenografts (59±3% ECN inhibition). Individual HDACs inhibited by SFN and ECN include HDACs 1, 2, 4 and 6. Interestingly, global acetylation status of histones H3 or H4 remain unaltered. The interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status is partially explained by decreased histone acetyl transferase activity (48.8±5.3%). In contrast, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1β and PP2A activity. Together, these findings suggest that ITCs modulate histone status via HDAC inhibition and phosphatase enhancement. This allows for reduced levels of histone H1 phosphorylation, a marker correlated with human bladder cancer progression. Therefore, ITC-mediated inhibition of histone H1 phosphorylation presents a novel direction of research in elucidating epidemiological relationships and supports future food-based prevention strategies.

Significance: Collectively, our findings suggest that the cruciferous vegetable isothiocyanates: sulforaphane (SFN) and erucin (ECN), impact histones status in bladder cancer cells by modulating specific HDACs and HATs, and enhancing phosphatase activity, resulting in reduction of histone H1 phosphorylation. These findings are significant due to the fact that our previous work positively correlated histone H1 phosphorylation with bladder cancer carcinogenesis and progression. Therefore, we propose that SFN and ECN may inhibit bladder carcinogenesis via epigenetic modulation of gene expression associated with histone H1 phosphorylation. These efforts may elucidate biomarkers useful in epidemiologic studies related to cruciferous vegetable intake and cancer risk or provide intermediate biomarkers for food-based clinical intervention studies in high-risk cohorts.
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http://dx.doi.org/10.1016/j.jprot.2017.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324139PMC
March 2017

Proteomic Signatures of Thymomas.

PLoS One 2016 10;11(11):e0166494. Epub 2016 Nov 10.

Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, OH, United States of America.

Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS) of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104371PMC
June 2017

Tag-Count Analysis of Large-Scale Proteomic Data.

J Proteome Res 2016 12 10;15(12):4742-4746. Epub 2016 Nov 10.

The Ohio State Biochemistry Graduate Program, The Ohio State University , Columbus, Ohio 43210, United States.

Label-free quantitative methods are advantageous in bottom-up (shotgun) proteomics because they are robust and can easily be applied to different workflows without additional cost. Both label-based and label-free approaches are routinely applied to discovery-based proteomics experiments and are widely accepted as semiquantitative. Label-free quantitation approaches are segregated into two distinct approaches: peak-abundance-based approaches and spectral counting (SpC). Peak abundance approaches like MaxLFQ, which is integrated into the MaxQuant environment, require precursor peak alignment that is computationally intensive and cannot be routinely applied to low-resolution data. Not limited by these constraints, SpC approaches simply use the number of peptide identifications corresponding to a given protein as a measurement of protein abundance. We show here that spectral counts from multidimensional proteomic data sets have a mean-dispersion relationship that can be modeled in edgeR. Furthermore, by simulating spectral counts, we show that this approach can routinely be applied to large-scale discovery proteomics data sets to determine differential protein expression.
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http://dx.doi.org/10.1021/acs.jproteome.6b00554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980212PMC
December 2016

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability.

BMC Biochem 2016 Oct 1;17(1):18. Epub 2016 Oct 1.

Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, 43210, USA.

Background: There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions.

Results: We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability.

Conclusion: Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.
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http://dx.doi.org/10.1186/s12858-016-0074-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045598PMC
October 2016

A multi-model statistical approach for proteomic spectral count quantitation.

J Proteomics 2016 07 31;144:23-32. Epub 2016 May 31.

The Ohio State Biochemistry Graduate Program, The Ohio State University, Columbus, OH, USA; Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA. Electronic address:

Unlabelled: The rapid development of mass spectrometry (MS) technologies has solidified shotgun proteomics as the most powerful analytical platform for large-scale proteome interrogation. The ability to map and determine differential expression profiles of the entire proteome is the ultimate goal of shotgun proteomics. Label-free quantitation has proven to be a valid approach for discovery shotgun proteomics, especially when sample is limited. Label-free spectral count quantitation is an approach analogous to RNA sequencing whereby count data is used to determine differential expression. Here we show that statistical approaches developed to evaluate differential expression in RNA sequencing experiments can be applied to detect differential protein expression in label-free discovery proteomics. This approach, termed MultiSpec, utilizes open-source statistical platforms; namely edgeR, DESeq and baySeq, to statistically select protein candidates for further investigation. Furthermore, to remove bias associated with a single statistical approach a single ranked list of differentially expressed proteins is assembled by comparing edgeR and DESeq q-values directly with the false discovery rate (FDR) calculated by baySeq. This statistical approach is then extended when applied to spectral count data derived from multiple proteomic pipelines. The individual statistical results from multiple proteomic pipelines are integrated and cross-validated by means of collapsing protein groups.

Biological Significance: Spectral count data from shotgun proteomics experiments is semi-quantitative and semi-random, yet a robust way to estimate protein concentration. Tag-count approaches are routinely used to analyze RNA sequencing data sets. This approach, termed MultiSpec, utilizes multiple tag-count based statistical tests to determine differential protein expression from spectral counts. The statistical results from these tag-count approaches are combined in order to reach a final MultiSpec q-value to re-rank protein candidates. This re-ranking procedure is completed to remove bias associated with a single approach in order to better understand the true proteomic differences driving the biology in question. The MultiSpec approach can be extended to multiple proteomic pipelines. In such an instance, MultiSpec statistical results are integrated by collapsing protein groups across proteomic pipelines to provide a single ranked list of differentially expressed proteins. This integration mechanism is seamlessly integrated with the statistical analysis and provides the means to cross-validate protein inferences from multiple proteomic pipelines.
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http://dx.doi.org/10.1016/j.jprot.2016.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967010PMC
July 2016

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.

J Proteomics 2016 Mar 13;136:89-98. Epub 2016 Jan 13.

Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.

Unlabelled: Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers.

Biological Significance: Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.
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http://dx.doi.org/10.1016/j.jprot.2015.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783258PMC
March 2016

The potential diagnostic power of extracellular vesicle analysis for multiple myeloma.

Expert Rev Mol Diagn 2016 28;16(3):277-84. Epub 2016 Jan 28.

c Department of Internal Medicine, Division of Hematology , The Ohio State University , Columbus , OH , USA.

Multiple myeloma (MM) is a hematologic malignancy of plasma cells (PCs). In the United States, MM accounts for approximately 1% of all diagnoses and 2% of all cancer-related deaths. Although MM is a treatable disease, most patients eventually relapse, and despite the development of numerous treatment options it is still considered incurable. Mechanisms of communication between MM-PCs and bone marrow microenvironment, including cell-cell contacts and release of pro-survival factors, promote cancer cell survival and drug resistance. Recently, the importance of extracellular vesicles (EVs) as mechanisms of communication between MM cells and other cells in the microenvironment has been reported. In this review, the authors provide the update on the biology and clinical aspects of EVs in MM.
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http://dx.doi.org/10.1586/14737159.2016.1132627DOI Listing
November 2016

Proteomic profiling identifies specific histone species associated with leukemic and cancer cells.

Clin Proteomics 2015 27;12(1):22. Epub 2015 Aug 27.

Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43210 USA.

Background: Chromatin is an extraordinarily complex structure. Much of this complexity results from the presence of numerous histone post-translational modifications and histone variants. Alterations in the patterns of histone post-translational modifications are emerging as a feature of many types of cancer and have been shown to have prognostic value.

Results: We have applied a liquid chromatography/mass spectrometry-based approach to comprehensively characterize the histone proteome in primary samples from chronic lymphocytic leukemia (CLL) patients, as well as bladder and breast cancer cell culture models. When compared to non-malignant CD19+ B cells from healthy donors, the CLL histone proteome showed a distinct signature of differentially expressed species, spanning all the histones studied and including both post-translationally modified species and unmodified, non-allelic replication-dependent histone isoforms. However, the large changes in histone H3 and H4 that are characteristic of many cancer types were not observed. One of species of H2A (mass = 14,063 Da) was the most strongly associated with time to treatment in CLL patients. CLL patient samples also demonstrated histone profiles that were distinct from those of the bladder and breast cancer cells.

Conclusions: Signatures of histone profiles are complex and can distinguish between healthy individuals and CLL patients and may provide prognostic markers. In addition, histone profiles may define tissue specific malignancies.
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http://dx.doi.org/10.1186/s12014-015-9095-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551702PMC
August 2015

Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence.

Mol Cell Proteomics 2016 Mar 24;15(3):818-33. Epub 2015 Jul 24.

From the ‡Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, 32310;

Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.
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http://dx.doi.org/10.1074/mcp.M114.046441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813703PMC
March 2016

Spinal Muscular Atrophy Biomarker Measurements from Blood Samples in a Clinical Trial of Valproic Acid in Ambulatory Adults.

J Neuromuscul Dis 2015 Jun;2(2):119-130

Department of Molecular & Cellular Biochemistry, The Ohio State University Wexner Medical Center, Columbus, OH, USA.

Background: Clinical trials of therapies for spinal muscular atrophy (SMA) that are designed to increase the expression the SMN protein ideally include careful assessment of relevant SMN biomarkers.

Objective: In the SMA VALIANT trial, a recent double-blind placebo-controlled crossover study of valproic acid (VPA) in ambulatory adult subjects with SMA, we investigated relevant pharmacodynamic biomarkers in blood samples from SMA subjects by direct longitudinal measurement of histone acetylation and SMN mRNA and protein levels in the presence and absence of VPA treatment.

Methods: Thirty-three subjects were randomized to either VPA or placebo for the first 6 months followed by crossover to the opposite arm for an additional 6 months. Outcome measures were compared between the two treatments (VPA and placebo) using a standard crossover analysis.

Results: A significant increase in histone H4 acetylation was observed with VPA treatment (p = 0.005). There was insufficient evidence to suggest a treatment effect with either full length or truncated SMN mRNA transcript levels or SMN protein levels.

Conclusions: These measures were consistent with the observed lack of change in the primary clinical outcome measure in the VALIANT trial. These results also highlight the added benefit of molecular and pharmacodynamic biomarker measurements in the interpretation of clinical trial outcomes.
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http://dx.doi.org/10.3233/JND-150081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5271431PMC
June 2015

The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Nucleic Acids Res 2015 Apr 23;43(7):3605-13. Epub 2015 Mar 23.

Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA

Early steps of gene expression are a composite of promoter recognition, promoter activation, RNA synthesis and RNA processing, and it is known that SUMOylation, a post-translational modification, is involved in transcription regulation. We previously found that SUMO-1 marks chromatin at the proximal promoter regions of some of the most active housekeeping genes during interphase in human cells, but the SUMOylated targets on the chromatin remained unclear. In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing. Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected. This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.
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http://dx.doi.org/10.1093/nar/gkv246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402547PMC
April 2015