Biochemistry Commons^™

Evidence Of A Perilipin 5 Splice Variant, Brodie Ranzau

Undergraduate Honors Thesis Projects

Alternative splicing occurs throughout the human genome, leading to multiple proteins from a single gene. The resulting proteins can be nearly identical or vastly different in how they function. Alternative splicing within the perilipin family has been observed in perilipins 1 and 3, giving rise to proteins with varying functions. In the course of our studies on perilipin 5, an immunoblot signal was observed that corresponded to an approximately 35 kDa protein. Western blot analysis of this protein has revealed its expression in C2C12 cultured myoblasts and in heart and liver mouse tissue. Further analysis of perilipin 5 cDNA through …

Expression, Purification And Characterization Of Lysine Methyltransferase Smyd5, Wen Xue

Wayne State University Theses

Methylation of histones and non-histone proteins play vital roles in numerous cellular processes including gene expression regulation and DNA damage response. The identifications of methyltransferase SMYD protein family are not well characterized. SMYD5 is a unique and critical member of SMYD family involved in immune response, stem cell renew, hematopoiesis regulation and cancer metastasis. Understanding its function and structure is monumental to human disease. With the achievement of SMYD5 expression and purification, the association between SMYD5 and its substrate histone H4 has been investigated. While possessing a poly-E tail instead of the TPR domain included in other members, SMYD5 is …

Frontiers In Otolith Chemistry: Insights, Advances And Applications, B. D. Walther, K. E. Limburg, C. M. Jones, J. J. Schaffler

OES Faculty Publications

The rapid proliferation of publications employing chemical assays of fish hard parts, and otoliths in particular, has led to many novel insights into the migration patterns, life history strategies and mixed stock dynamics of fishes across the globe (Campana et al., 2000; Elsdon et al., 2008; Walther & Limburg, 2012). These insights include uncovering diverse migratory strategies within species and populations (Kerr et al., 2009; Hogan et al., 2014; Schoen et al., 2016), quantifying rates of mixing among stocks across management boundaries (Rooker et al., 2008; Walther & Thorrold, 2010) and estimating the relative …

Beyond Zar: The Use And Abuse Of Classification Statistics For Otolith Chemistry, C. M. Jones, M. Palmers, J. J. Schaffler

OES Faculty Publications

Classification method performance was evaluated using otolith chemistry of juvenile Atlantic menhaden Brevoortia tyrannus when assumptions of data normality were met and were violated. Four methods were tested [linear discriminant function analysis (LDFA), quadratic discriminant function analysis (QDFA), random forest (RF) and artificial neural networks (ANN)] using computer simulation to determine their performance when variable-group means ranged from small to large and their performance under conditions of typical skewness to double the amount of skewness typically observed. Using the kappa index, the parametric methods performed best after applying appropriate data transformation, gaining 2% better performance with LDFA performing slightly better …

Studies Of Sumoylation In Regulating Mif Stability And Rangap1 Nucleo-Cytoplasmic Shuttling In Controlling Its Sumo Modification, Progga Sen

Wayne State University Dissertations

SUMOylation is an essential post-translational modification that regulates a variety of critical cellular pathways ranging from nuclear transport to protein stability. Accumulating lines of evidence have shown that a perturbation of the SUMOylation pathway is associated with human diseases, especially various types of cancer. Our recent proteomic studies revealed a drastic increase in levels of SUMO2/3 modification on the proinflammatory cytokine MIF in the metastatic breast cancer cell line compared to the non-metastatic control cell line. Interestingly, the increase in levels of both MIF and global SUMO-2/3 modification in the metastatic cells are positively correlated to that of unmodified MIF …

Nitrosative Stress Sensing In Porphyromonas Gingivalis: Structure And Function Of The Heme Binding Transcriptional Regulator Hcpr, Benjamin R. Belvin

Theses and Dissertations

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of …

Comparing An Atomic Model Or Structure To A Corresponding Cryo-Electron Microscopy Image At The Central Axis Of A Helix, Stephanie Zeil, Julio Kovacs, Willy Wriggers, Jing He

Computer Science Faculty Publications

Three-dimensional density maps of biological specimens from cryo-electron microscopy (cryo-EM) can be interpreted in the form of atomic models that are modeled into the density, or they can be compared to known atomic structures. When the central axis of a helix is detectable in a cryo-EM density map, it is possible to quantify the agreement between this central axis and a central axis calculated from the atomic model or structure. We propose a novel arc-length association method to compare the two axes reliably. This method was applied to 79 helices in simulated density maps and six case studies using cryo-EM …

An Effective Computational Method Incorporating Multiple Secondary Structure Predictions In Topology Determination For Cryo-Em Images, Abhishek Biswas, Desh Ranjan, Mohammad Zubair, Stephanie Zeil, Kamal Al Nasr, Jing He

Computer Science Faculty Publications

A key idea in de novo modeling of a medium-resolution density image obtained from cryo-electron microscopy is to compute the optimal mapping between the secondary structure traces observed in the density image and those predicted on the protein sequence. When secondary structures are not determined precisely, either from the image or from the amino acid sequence of the protein, the computational problem becomes more complex. We present an efficient method that addresses the secondary structure placement problem in presence of multiple secondary structure predictions and computes the optimal mapping. We tested the method using 12 simulated images from alpha-proteins and …

Exosomes Serve As Novel Modes Of Tick-Borne Flavivirus Transmission From Arthropod To Human Cells And Facilitates Dissemination Of Viral Rna And Proteins To The Vertebrate Neuronal Cells, Wenshuo Zhou, Michael Woodson, Biswas Neupane, Fengwei Bai, Michael B. Sherman, Kyung H. Choi, Girish Neelakanta, Hameeda Sultana

Biological Sciences Faculty Publications

Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes …

Metagomics: A Web-Based Tool For Peptide-Centric Functional And Taxonomic Analysis Of Metaproteomics Data, Michael Riffle, Damon H. May, Emma Timmins-Schiffman, Molly P. Mikan, Daniel Jaschob, William S. Noble, Brook L. Nunn

OES Faculty Publications

Metaproteomics is the characterization of all proteins being expressed by a community of organisms in a complex biological sample at a single point in time. Applications of metaproteomics range from the comparative analysis of environmental samples (such as ocean water and soil) to microbiome data from multicellular organisms (such as the human gut). Metaproteomics research is often focused on the quantitative functional makeup of the metaproteome and which organisms are making those proteins. That is: What are the functions of the currently expressed proteins? How much of the metaproteome is associated with those functions? And, which microorganisms are expressing the …

Portulaca Oleracea Extract Can Inhibit Nodule Formation Of Colon Cancer Stem Cells By Regulating Gene Expression Of Notch Signal Transduction Pathway, Li Chen

Dissertations, Master's Theses and Master's Reports

The main aim of this study was to investigate the effects of Portulaca oleracea extract on tumor formation in colon cancer stem cells and chemotherapy sensitivity. In addition, this study analyzed the genetic changes within the Notch signal transduction pathway associated with the effects of the extracts. Serum-free cultures of colon cancer cells (HT-29) and HT-29 stem cells were treated with the chemotherapeutic drug 5-FU to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the …

Heterologous Expression And Purification Of Full-Length Human Polybromo-1 Protein, Sarah Hopson

Dissertations, Master's Theses and Master's Reports

Over the past decade, it has become apparent that the human polybromo-1 protein (BAF180) has a critical role in cancer. BAF180 is known to be a driver mutation in clear cell renal cell carcinoma, where it has been found to be mutated in approximately 40% of cases. Mutations have also been found in several other cancers, including intrahepatic cholangiocarcinomas and epithelioid sarcomas. BAF180 is the chromatin targeting subunit of the PBAF (Polybromo-associated BRG1-associated factor) chromatin remodeling complex, a role facilitated by its nine domains: six bromodomains, which recognize and bind to acetylated lysines on histones; two BAH (bromo-adjacent homology) domains, …

The Maculoprotective Effect Of A Thiol Antioxidant In Retinal Degeneration Models, Hsiu-Jen Wang

Doctoral Dissertations

"Age-related macular degeneration (AMD) is a leading cause of irreversible blindness among adults, age 60 and older, in developed countries. While oxidative stress is implicated in the pathogenesis of AMD, clinical studies have shown that dietary antioxidants can delay progression of AMD. Currently, there is no FDA-approved treatment for AMD. Therefore, we hypothesized that N-acetylcysteine amide (NACA), a thiol antioxidant, would protect retinal pigment epithelium and impede the progression of retinal degeneration. The goal of this work was to evaluate the efficacy of NACA in preventing retinal pigment epithelial cell and photoreceptor death in AMD models. To achieve this, we …

การวิเคราะห์รูปแบบการแสดงออกของโปรตีโอมและอินเทอแรกโตมเพื่อค้นหาสารบ่งชี้สำหรับโรคออทิซึมสเปกตรัม, ฉัตรระวี พิชิตพันธ์พงศ์

Chulalongkorn University Theses and Dissertations (Chula ETD)

ในปัจจุบัน ออทิซึมสเปกตรัม (Autism spectrum disorder, ASD) ยังคงเป็นโรคที่ยังคงไม่ทราบกลไกระดับโมเลกุล แน่ชัด ยีนและโปรตีนที่เคยมีการรายงานการแสดงออกผิดปกติไปในผู้ป่วยออทิซึมสเปกตรัมมีความสามารถในการทำซ้ำ (Reproducibility) ต่ำหรืออาจไม่มีเลย ซึ่งน่าจะมาจากความหลากหลายของอาการแสดงของผู้ป่วย จากการศึกษาล่าสุด แสดงให้เห็นว่าการจัดกลุ่มออทิซึมสเปกตรัมออกเป็นกลุ่มย่อยตามความรุนแรงของลักษณะทางคลินิกสามารถช่วยในการระบุยีนและกลไกที่ผิดปกติไปในกลุ่มย่อยของผู้ป่วย อย่างไรก็ตาม การแบ่งกลุ่มผู้ป่วยเป็นกลุ่มย่อยตามความรุนแรงของลักษณะทางคลินิกใน ออทิซึมสเปกตรัมยังไม่มีการใช้กันอย่างแพร่หลายในการค้นหาโปรตีน Biomarker ที่เกี่ยวข้องกับออทิซึมสเปกตรัม ในการศึกษาครั้งนี้ ผู้วิจัยทำการวิเคราะห์โดยการแบ่งกลุ่มผู้ป่วยจำนวน 85 รายออกเป็นกลุ่มย่อยทางคลินิกตามคะแนน ADI-R และทำการวิเคราะห์รูปแบบการแสดงออกของยีนในออทิซึมสเปกตรัมในแต่ละกลุ่มย่อยเปรียบเทียบกับกลุ่มควบคุมคนปกติที่อายุใกล้เคียงกันและเพศเดียวกันกับผู้ป่วย (Sex/age-matched control) ซึ่งสามารถระบุยีนที่มีการแสดงออกผิดปกติ (Differentially expressed genes, DEGs) ในออทิซึมสเปกตรัม นอกจากนี้ ผู้วิจัยได้ทำการวิเคราะห์รูปแบบการแสดงออกของโปรตีโอมจากเซลล์ไลน์เม็ดเลือดขาว (Lymphoblastoid cell lines,LCLs) ด้วยเทคนิค Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) ร่วมกับ Nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) รายชื่อของโปรตีนที่มีการแสดงออกผิดปกติไปอย่างมีนัยสำคัญที่ได้จากการวิเคราะห์ถูกนำมาเปรียบเทียบกับ DEGs และผลการวิเคราะห์ Meta-analysis ของโปรตีนและโปรตีโอมในการศึกษาก่อนหน้า หน้าที่ทางพยาธิสภาพที่มีความเกี่ยวข้องกับโปรตีนถูกวิเคราะห์โดยโปรแกรม Ingenuity Pathway Analysis (IPA) และได้ทำการคัดเลือกโปรตีนที่น่าสนใจมาทำการตรวจสอบโดยใช้ Western blot analysis จากการศึกษาครั้งนี้พบว่าผลการวิเคราะห์การแบ่งกลุ่มผู้ป่วยออกเป็นกลุ่มย่อยทางคลินิกตามคะแนน ADI-R สามารถแบ่งผู้ป่วยออกเป็น 4 กลุ่มย่อย ซึ่งพบว่าหนึ่งในกลุ่มนั้น เป็นกลุ่มออทิซึมสเปกตรัมที่มีความบกพร่องทางภาษารุนแรง จากผลวิเคราะห์รูปแบบ ทรานสคริปโตมของเซลล์ไลน์เม็ดเลือดขาว พบว่ามีจุดโปรตีนจำนวน 82 จุดที่มีการแสดงออกผิดปกติไปอย่างมีนัยสำคัญในกลุ่มออทิซึมสเปกตรัมที่มีความบกพร่องทางภาษารุนแรง และได้ทำการคัดเลือก 18 จุดโปรตีนจากทั้งหมดมาวิเคราะห์ชนิดของโปรตีนโดยเทคนิค Nano-LC-MS/MS ซึ่งพบว่ามีความเกี่ยวข้องกับการทำงานของระบบประสาทและกระบวนการอักเสบ จากการศึกษานี้พบว่าจากโปรตีนทั้งหมด การแสดงออกของ Isocitrate dehydrogenase (IDH2) และ Diazepam binding inhibitor (DBI) ลดลงในออทิซึมสเปกตรัม ซึ่งโปรตีนเหล่านี้ยังพบการแสดงออกผิดปกติในการวิเคราะห์ Meta-analysis ของทรานสคริปโตมและโปรตีโอมร่วมด้วย การแบ่งกลุ่มออทิซึมสเปกตรัมออกเป็นกลุ่มย่อยทางคลินิกตามด้วยการวิเคราะห์ทรานสคริปโตมและโปรตีโอมแบบบูรณาการร่วมกัน ทำให้การศึกษานี้สามารถระบุโปรตีนที่มีการแสดงออกผิดปกติไปอย่างมีนัยสำคัญในกลุ่มย่อยของออทิซึมสเปกตรัมที่มีความบกพร่องทางภาษาอย่างรุนแรง ซึ่งโปรตีนเหล่านี้อาจทำหน้าที่เป็นตัวแทนในการช่วยระบุกลไกทางโมเลกุลของออทิซึมสเปกตรัมในอนาคต ตลอดจน อาจพัฒนาไปเป็น …

Characterization Of Quorum-Quenching Lactonases And Plp-Dependent Aminotransferases: Structure, Mechanism And Alternative Turnover, Romila Nina Mascarenhas

Dissertations

Cell-to-cell communication by bacteria is essential for the regulation of gene expression important in colonization, biofilm formation, virulence and other processes. This communication is called "quorum-sensing" and is mediated by small molecules called autoinducers. One major class of autoinducers used by gram-negative bacteria is N-acyl homoserine lactones (AHL's). Enzymes capable of disrupting this communication are called "quorum-quenching" catalysts and have proven to be invaluable biochemical tools for understanding quorum-sensing pathways. Quorum-quenching enzymes hold promise for application in anti-biofouling, agriculture, aquaculture, bioremediation and other synthetic biology settings. However, the mechanisms that these enzymes use to recognize and process their substrates are …

Evolution Of The Kinetics And Dynamics Of Heme-Crevice Loop Regulating Chemistry In Human Cytochrome C, Shiloh M. Nold

Graduate Student Theses, Dissertations, & Professional Papers

Cytochrome c, cytc, is a metalloprotein that plays primary roles in electron transport and intrinsic apoptotic pathways. Much of the chemistry that cytc is involved with is regulated by a highly conserved region known as the heme crevice loop, consisting of residues 70-85. Only three of these residues (those at positions 81, 83 and 85) are not universally conserved within the evolutionary timeline. Here I look to elucidate possible evolutionary roles for several of the key residues known to be important in regulating heme chemistry of cytc.

I first address the role that lysine …

The Human Mitochondrial Chaperonin: It Takes Two Single-Rings To Tango, Adrian Sergio Enriquez

Open Access Theses & Dissertations

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding and assembly of newly imported mitochondrial proteins into their biologically active state. It is composed of two proteins from the highly conserved heat shock protein family, hsp10 and hsp60, that assemble into large oligomeric complexes responsible for mediating the folding of non-native polypeptides in an ATP dependent manner. In addition to its innate role in protein folding, human mitochondrial hsp60 has been implicated in numerous moonlighting cellular activities that have been linked to diseases conditions such as cancer and neurodegeneracy. In light of its cellular importance, the …

New Open Conformation Of Smyd3 Implicates Conformational Selection And Allostery, Nicholas Spellmon, Xiaonan Sun, Wen Xue, Joshua Holcomb, Srinivas Chakravarthy, Weifeng Shang, Brian Fp Edwards, Nualpun Sirinupong, Chunying Li, Zhe Yang

Biochemistry and Molecular Biology Faculty Publications

SMYD3 plays a key role in cancer cell viability, adhesion, migration and invasion. SMYD3 promotes formation of inducible regulatory T cells and is involved in reducing autoimmunity. However, the nearly “closed” substrate-binding site and poor in vitro H3K4 methyltransferase activity have obscured further understanding of this oncogenically related protein. Here we reveal that SMYD3 can adopt an “open” conformation using molecular dynamics simulation and small-angle X-ray scattering. This ligand-binding-capable open state is related to the crystal structure-like closed state by a striking clamshell-like inter-lobe dynamics. The two states are characterized by many distinct structural and dynamical differences and the conformational …

Defining The Role Of Phosphorylation And Dephosphorylation In The Regulation Of Gap Junction Proteins, Hanjun Li

Theses & Dissertations

Gap junctions are intercellular channels that permit the free passage of ions, small metabolites, and signaling molecules between neighboring cells. In the diseased human heart, altered ventricular gap junction organization and connexin expression (i.e., remodeling) are key contributors to rhythm disturbances and contractile dysfunction. Connexin43 (Cx43) is the dominant gap junction protein isoform in the ventricle which is under tight regulation by serine/tyrosine phosphorylation. Phosphorylation and dephosphorylation regulate many aspects of Cx43 function including trafficking, assembly and disassembly, electrical and metabolic coupling at the plaque, as well as to modulate the interaction with other proteins.

Serine phosphorylation has long been …

Dna Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent Of Error-Prone Synthesis, And The Role Of Dntp Pools, Olga V. Kochenova

Theses & Dissertations

Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. Cells utilize specialized low-fidelity Translesion Synthesis (TLS) DNA polymerases to bypass lesions and rescue arrested replication forks. TLS is a highly conserved two-step process that involves insertion of a nucleotide opposite a lesion and extension of the resulting aberrant primer terminus. The first step can be performed by both replicative and TLS DNA polymerases and, because of non-instructive DNA lesions, often results in a nucleotide misincorporation. The second step is almost exclusively catalyzed by DNA polymerase ζ …