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Full-Text Articles in Molecular Biology

Identification Of Novel Δnp63Α-Regulated Mirnas Using An Optimized Small Rna-Seq Analysis Pipeline, Suraj Sakaram, Michael P. Craig, Natasha T. Hill, Amjad Aljiagthmi, Christian Garrido, Oleg Paliy, Michael Bottomley, Michael L. Raymer, Madhavi Kadakia Jul 2018

Identification Of Novel Δnp63Α-Regulated Mirnas Using An Optimized Small Rna-Seq Analysis Pipeline, Suraj Sakaram, Michael P. Craig, Natasha T. Hill, Amjad Aljiagthmi, Christian Garrido, Oleg Paliy, Michael Bottomley, Michael L. Raymer, Madhavi Kadakia

Biochemistry and Molecular Biology Faculty Publications

Advances in high-throughput sequencing have enabled profiling of microRNAs (miRNAs), however, a consensus pipeline for sequencing of small RNAs has not been established. We built and optimized an analysis pipeline using Partek Flow, circumventing the need for analyzing data via scripting languages. Our analysis assessed the effect of alignment reference, normalization method, and statistical model choice on biological data. The pipeline was evaluated using sequencing data from HaCaT cells transfected with either a non-silencing control or siRNA against ΔNp63α, a p53 family member protein which is highly expressed in non-melanoma skin cancer and shown to regulate a number of miRNAs ...


Microrna Involvement In The Onset And Progression Of Barrett’S Esophagus: A Systematic Review, Reilly J. Clark, Michael P. Craig, Sangeeta Agrawal, Madhavi Kadakia Jan 2018

Microrna Involvement In The Onset And Progression Of Barrett’S Esophagus: A Systematic Review, Reilly J. Clark, Michael P. Craig, Sangeeta Agrawal, Madhavi Kadakia

Biochemistry and Molecular Biology Faculty Publications

Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that develops from Barrett's esophagus (BE), an intestinal metaplasia of the distal esophagus. microRNAs (miRNAs), short non-coding regulatory RNAs, are frequently dysregulated in BE and are thought to play key roles in the onset of BE and its progression to EAC. miRNAs thus have potential diagnostic and prognostic value and are increasingly being used as cancer biomarkers. This review summarizes the current literature related to miRNAs that are dysregulated in BE within the context of Hedgehog, Notch, MAPK, NF kappa-B, Wnt and epithelial-mesenchymal transition (EMT) signaling which are thought to drive ...


Δnp63Α And Microrna: Leveraging The Epithelial-Mesenchymal Transition, Andrew J. Stacy, Michael P. Craig, Suraj Sakaram, Madhavi Kadakia Jan 2017

Δnp63Α And Microrna: Leveraging The Epithelial-Mesenchymal Transition, Andrew J. Stacy, Michael P. Craig, Suraj Sakaram, Madhavi Kadakia

Biochemistry and Molecular Biology Faculty Publications

The epithelial-mesenchymal transition (EMT) is a cellular reprogramming mechanism that is an underlying cause of cancer metastasis. Recent investigations have uncovered an intricate network of regulation involving the TGFβ Wnt, and Notch signaling pathways and small regulatory RNA species called microRNAs (miRNAs). The activity of a transcription factor vital to the maintenance of epithelial stemness, ?Np63a, has been shown to modulate the activity of these EMT pathways to either repress or promote EMT. Furthermore, ?Np63a is a known regulator of miRNA, including those directly involved in EMT. This review discusses the evidence of ?Np63a as a master regulator of EMT ...


Alignment Of Mitotic Chromosomes In Human Cells Involves Sr-Like Splicing Factors Btf And Trap150, Sapna Varia, Divya Cheedu, Michael P. Markey, Keshia Torres-Shafer, Vishnu P. Battini, Athanasios Bubulya, Paula A. Bubulya Jan 2017

Alignment Of Mitotic Chromosomes In Human Cells Involves Sr-Like Splicing Factors Btf And Trap150, Sapna Varia, Divya Cheedu, Michael P. Markey, Keshia Torres-Shafer, Vishnu P. Battini, Athanasios Bubulya, Paula A. Bubulya

Biochemistry and Molecular Biology Faculty Publications

Serine-arginine-rich (SR) or SR-like splicing factors interact with exon junction complex proteins during pre-mRNA processing to promote mRNA packaging into mature messenger ribonucleoproteins (mRNPs) and to dictate mRNA stability, nuclear export, and translation. The SR protein family is complex, and while many classical SR proteins have well-defined mRNA processing functions, those of other SR-like proteins is unclear. Here, we show that depletion of the homologous non-classical serine-arginine-rich (SR) splicing factors Bcl2-associated transcription factor (Btf or BCLAF) and thyroid hormone receptor-associated protein of 150 kDa (TRAP150) causes mitotic defects. We hypothesized that the depletion of these SR-like factors affects mitosis indirectly ...


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 Dec 2016

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 ...


Pld-Specific Small-Molecule Inhibitors Decrease Tumor-Associated Macrophages And Neutrophils Infiltration In Breast Tumors And Lung And Liver Metastases, Karen M. Henkels, Naveen Reddy Muppani, Julian Gomez-Cambronero Nov 2016

Pld-Specific Small-Molecule Inhibitors Decrease Tumor-Associated Macrophages And Neutrophils Infiltration In Breast Tumors And Lung And Liver Metastases, Karen M. Henkels, Naveen Reddy Muppani, Julian Gomez-Cambronero

Biochemistry and Molecular Biology Faculty Publications

Phospholipase D-2 (PLD2) has a key role in breast cancer formation and metastasis formation with PLD small inhibitors reducing primary tumor growth. This study aimed to evaluate the importance of targeting PLD on the tumor microenvironment. We provide evidence about the beneficial effect of PLD inhibitors [FIPI (dual PLD1/PLD2) or VU0155072-2 (PLD2 inhibitor)] on avoiding infiltration of tumor-helping macrophages and neutrophils. Tumor growth and metastasis within the primary tumors had low (<20% over controls) PLD enzyme activity. Unexpectedly, we found that the inhibitors also affected PLD2 gene expression and protein albeit at a lesser extent. The later could indicate that targeting both the actual PLD enzyme and its activity could be beneficial for potential cancer treatments in vivo. F4/80 and Ly6G staining of macrophages and neutrophils, respectively, and Arg1 staining data were consistent with M2 and N2 polarization. NOS2 staining increased in xenotransplants ...


Copy Number Variation In Archival Melanoma Biopsies Versus Benign Melanocytic Lesions, Ahmed Mahas, Keerti Potluri, Michael N. Kent, Sameep Naik, Michael P. Markey Jan 2016

Copy Number Variation In Archival Melanoma Biopsies Versus Benign Melanocytic Lesions, Ahmed Mahas, Keerti Potluri, Michael N. Kent, Sameep Naik, Michael P. Markey

Biochemistry and Molecular Biology Faculty Publications

BACKGROUND: Skin melanocytes can give rise to benign and malignant neoplasms. Discrimination of an early melanoma from an unusual/atypical benign nevus can represent a significant challenge. However, previous studies have shown that in contrast to benign nevi, melanoma demonstrates pervasive chromosomal aberrations. OBJECTIVE: This substantial difference between melanoma and benign nevi can be exploited to discriminate between melanoma and benign nevi. METHODS: Array-comparative genomic hybridization (aCGH) is an approach that can be used on DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues to assess the entire genome for the presence of changes in DNA copy number. In this study, high ...


Myocardium And Bmp Signaling Are Required For Endocardial Differentiation, Sharina Palencia-Desai, Megan S. Rost, Jennifer A. Schumancher, Quynh V. Ton, Michael P. Craig, Kristina Baltrunaite, Andrew L. Koenig, Jinhu Wang, Kenneth D. Poss, Neil C. Chi, Didier Y.R. Stainier Jan 2015

Myocardium And Bmp Signaling Are Required For Endocardial Differentiation, Sharina Palencia-Desai, Megan S. Rost, Jennifer A. Schumancher, Quynh V. Ton, Michael P. Craig, Kristina Baltrunaite, Andrew L. Koenig, Jinhu Wang, Kenneth D. Poss, Neil C. Chi, Didier Y.R. Stainier

Biochemistry and Molecular Biology Faculty Publications

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation ...


Role Of Vitamin D3 In Modulation Of Δnp63Α Expression During Uvb Induced Tumor Formation In Skh-1 Mice, Natasha Tremayne Hill, Gabriel H. Gracia-Maldondo, Mary K. Leonard, Amanda R. Harper, Kathleen L. Tober, Tatiana M. Oberyszyn, Madhavi P. Kadakia Sep 2014

Role Of Vitamin D3 In Modulation Of Δnp63Α Expression During Uvb Induced Tumor Formation In Skh-1 Mice, Natasha Tremayne Hill, Gabriel H. Gracia-Maldondo, Mary K. Leonard, Amanda R. Harper, Kathleen L. Tober, Tatiana M. Oberyszyn, Madhavi P. Kadakia

Biochemistry and Molecular Biology Faculty Publications

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D3 protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D3 and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing ...


Serdemetan Antagonizes The Mdm2-Hif1Α Axis Leading To Decreased Levels Of Glycolytic Enzymes, Jason Alexander Lehman, Paula M. Hauck, Jaimie M. Gendron, Christopher N. Batuello, Jacob A. Eitel, Allan Albig, Madhavi P. Kadakia, Lindsey D. Mayo Sep 2013

Serdemetan Antagonizes The Mdm2-Hif1Α Axis Leading To Decreased Levels Of Glycolytic Enzymes, Jason Alexander Lehman, Paula M. Hauck, Jaimie M. Gendron, Christopher N. Batuello, Jacob A. Eitel, Allan Albig, Madhavi P. Kadakia, Lindsey D. Mayo

Biochemistry and Molecular Biology Faculty Publications

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes ...


Sarcoptes Scabiei Mites Modulate Gene Expression In Human Skin Equivalents, Marjorie S. Morgan, Larry G. Arlian, Michael P. Markey Aug 2013

Sarcoptes Scabiei Mites Modulate Gene Expression In Human Skin Equivalents, Marjorie S. Morgan, Larry G. Arlian, Michael P. Markey

Biochemistry and Molecular Biology Faculty Publications

The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25 ...


Microrna-34a Modulates Mdm4 Expression Via A Target Site In The Open Reading Frame, Pooja Mandke, Nicholas Wyatt, Jillian Fraser, Benjamin Bates, Steven J. Berberich, Michael P. Markey Aug 2012

Microrna-34a Modulates Mdm4 Expression Via A Target Site In The Open Reading Frame, Pooja Mandke, Nicholas Wyatt, Jillian Fraser, Benjamin Bates, Steven J. Berberich, Michael P. Markey

Biochemistry and Molecular Biology Faculty Publications

Background

MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt ...


Son Maintains Accurate Splicing For A Subset Of Human Pre-Mrnas, Alok Sharma, Michael P. Markey, Keshia Torres-Munoz, Sapna Varia, Madhavi P. Kadakia, Athanasios Bubulya, Paula A. Bubulya Dec 2011

Son Maintains Accurate Splicing For A Subset Of Human Pre-Mrnas, Alok Sharma, Michael P. Markey, Keshia Torres-Munoz, Sapna Varia, Madhavi P. Kadakia, Athanasios Bubulya, Paula A. Bubulya

Biochemistry and Molecular Biology Faculty Publications

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed ...


Unsupplemented Artemia Diet Results In Reduced Growth And Jaw Dysmorphogenesis In Zebrafish, Michael P. Craig, Mitul B. Desai, Kate E. Olukalns, Scott E. Afton, Joseph A. Caruso, Jay R. Hove Jan 2011

Unsupplemented Artemia Diet Results In Reduced Growth And Jaw Dysmorphogenesis In Zebrafish, Michael P. Craig, Mitul B. Desai, Kate E. Olukalns, Scott E. Afton, Joseph A. Caruso, Jay R. Hove

Biochemistry and Molecular Biology Faculty Publications

The number of laboratories using zebrafish as an experimental animal model has risen tremendously over the past two decades (Craig et al., 2006). As a result, the number of zebrafish facilities around the world has dramatically increased to meet the elevated demand for proper animal care and maintenance. In order to meet this demand, aquaculture facilities must employ husbandry protocols designed to produce a constant supply of healthy, viable eggs. Surprisingly, many husbandry strategies, particularly feeding protocols, are frequently passed down from members of one lab to another in a colloquial fashion without rigorous experimental validation. An ideal diet should ...


Resonance Assignments And Secondary Structure Predictions Of The As(Iii) Metallochaperone Arsd In Solution, Jun Ye, Yanan He, Jack Skalicky, Barry P. Rosen, Timothy L. Stemmler Nov 2010

Resonance Assignments And Secondary Structure Predictions Of The As(Iii) Metallochaperone Arsd In Solution, Jun Ye, Yanan He, Jack Skalicky, Barry P. Rosen, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear. We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA. The predicted solution structure of ArsD is in agreement with ...


Frataxin And Mitochondrial Fes Cluster Biogenesis, Timothy L. Stemmler, Emmanuel Lesuisse, Debumar Pain, Andrew Dancis Aug 2010

Frataxin And Mitochondrial Fes Cluster Biogenesis, Timothy L. Stemmler, Emmanuel Lesuisse, Debumar Pain, Andrew Dancis

Biochemistry and Molecular Biology Faculty Publications

Friedreich’s ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in Fe-S cluster assembly in mitochondria. Fe-S clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multi-step and multi-subunit mitochondrial machinery that includes a scaffold protein Isu for assembling a protein bound Fe-S cluster intermediate. Frataxin interacts with Isu, iron, and with the cysteine desulfurase Nfs1 that supplies sulfur, thus placing it at the center of mitochondrial Fe-S cluster biosynthesis.


Oxidation Of Methane By A Biological Dicopper Centre, Ramakrishnan Balasubramanian, Stephen M. Smith, Swati Rawat, Liliya A. Yatsunyk, Timothy L. Stemmler, Amy C. Rosenzweig Apr 2010

Oxidation Of Methane By A Biological Dicopper Centre, Ramakrishnan Balasubramanian, Stephen M. Smith, Swati Rawat, Liliya A. Yatsunyk, Timothy L. Stemmler, Amy C. Rosenzweig

Biochemistry and Molecular Biology Faculty Publications

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol1. Current processes to activate the strong C–H bond (104 kcal mol−1) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst3. There are two types of MMO. Soluble MMO (sMMO),expressed by ...


Nmr Assignments Of A Stable Processing Intermediate Of Human Frataxin, Kalyan C. Kondapalli, Krisztina Z. Bencze, Eric Dizin, James A. Cowan, Timothy L. Stemmler Jan 2010

Nmr Assignments Of A Stable Processing Intermediate Of Human Frataxin, Kalyan C. Kondapalli, Krisztina Z. Bencze, Eric Dizin, James A. Cowan, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

Frataxin, a nuclear encoded protein targeted to the mitochondrial matrix, has recently been implicated as an iron chaperone that delivers ferrous iron to the iron-sulfur assembly enzyme IscU. During transport across the mitochondrial membrane, the N-terminal mitochondrial targeting sequence of frataxin is cleaved in a two-step process to produce the mature protein found in the matrix, however N-terminal extended forms of the protein have also been observed in vivo. The recent structural characterization studies of the human frataxin ortholog were performed on a truncated variant of the protein. Here we report the NMR spectral assignment of an extended form of ...


Self-Assembly And Disassembly Of The Snare Complex: Examined Using Circular Dichroism And Atomic Force Microscopy, Jeremy D. Cook, Won Jin Cho, Timothy L. Stemmler, Bhanu P. Jena Sep 2009

Self-Assembly And Disassembly Of The Snare Complex: Examined Using Circular Dichroism And Atomic Force Microscopy, Jeremy D. Cook, Won Jin Cho, Timothy L. Stemmler, Bhanu P. Jena

Biochemistry and Molecular Biology Faculty Publications

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.


Alterations In Gene Expression And Sensitivity To Genotoxic Stress Following Hdmx Or Hdm2 Knockdown In Human Tumor Cells Harboring Wild-Type P53, Katherine Heminger, Michael P. Markey, Meldrick Mpagi, Steven J. Berberich Jan 2009

Alterations In Gene Expression And Sensitivity To Genotoxic Stress Following Hdmx Or Hdm2 Knockdown In Human Tumor Cells Harboring Wild-Type P53, Katherine Heminger, Michael P. Markey, Meldrick Mpagi, Steven J. Berberich

Biochemistry and Molecular Biology Faculty Publications

While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth. Cell cycle and growth assays ...


Structure And Dynamics Of Metalloproteins In Live Cells, Jeremy D. Cook, James E. Penner-Hahn, Timothy L. Stemmler Dec 2008

Structure And Dynamics Of Metalloproteins In Live Cells, Jeremy D. Cook, James E. Penner-Hahn, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

X-ray absorption spectroscopy (XAS) has emerged as one of the premier tools for investigating the structure and dynamic properties of metals in cells and in metal containing biomolecules. Utilizing the high flux and broad energy range of X-rays supplied by synchrotron light sources, one can selectively excite core electronic transitions in each metal. Spectroscopic signals from these electronic transitions can be used to dissect the chemical architecture of metals in cells, in cellular components and in biomolecules at varying degrees of structural resolution. With the development of ever-brighter X-ray sources, X-ray methods have grown into applications that can be utilized ...


Evolution Of Metal(Loid) Binding Sites In Transcriptional Regulators, Efrén Ordóñez, Saravanamuthu Thiyagarajan, Jeremy D. Cook, Timothy L. Stemmler, José A. Gil., Luís M. Mateos, Barry P. Rosen Jun 2008

Evolution Of Metal(Loid) Binding Sites In Transcriptional Regulators, Efrén Ordóñez, Saravanamuthu Thiyagarajan, Jeremy D. Cook, Timothy L. Stemmler, José A. Gil., Luís M. Mateos, Barry P. Rosen

Biochemistry and Molecular Biology Faculty Publications

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II) and Ni(II). These homodimeric repressors bind to DNA in absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid ...


A Cytosolic Iron Chaperone That Delivers Iron To Ferritin, Haifeng Shi, Krisztina Z. Bencze, Timothy L. Stemmler, Caroline C. Philpott May 2008

A Cytosolic Iron Chaperone That Delivers Iron To Ferritin, Haifeng Shi, Krisztina Z. Bencze, Timothy L. Stemmler, Caroline C. Philpott

Biochemistry and Molecular Biology Faculty Publications

Ferritins are the main iron storage proteins found in animals, plants and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. The human Poly r(C)-Binding Protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo, and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools ...


Association Of Copper To Riboflavin Binding Protein; Characterization By Epr And Xas, Shelia R. Smith, Krisztina Z. Bencze, Kristen Wasiukanis, Timothy L. Stemmler, Marilee Benore-Parsons Jan 2008

Association Of Copper To Riboflavin Binding Protein; Characterization By Epr And Xas, Shelia R. Smith, Krisztina Z. Bencze, Kristen Wasiukanis, Timothy L. Stemmler, Marilee Benore-Parsons

Biochemistry and Molecular Biology Faculty Publications

The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies. The type II site contains a mix of copper I and II in an oxygen rich environment.


Characterization And Structure Of A Zn2+ And [2fe-2s]-Containing Copper Chaperone From Archaeoglobus Fulgidus, Matthew H. Sazinsky, Benjamin Lemoine, Maria Orofino, Roman Davydov, Krisztina Z. Bencze, Timothy L. Stemmler, Brian M. Hoffman, José M. Argüello, Amy C. Rosenzweig Jul 2007

Characterization And Structure Of A Zn2+ And [2fe-2s]-Containing Copper Chaperone From Archaeoglobus Fulgidus, Matthew H. Sazinsky, Benjamin Lemoine, Maria Orofino, Roman Davydov, Krisztina Z. Bencze, Timothy L. Stemmler, Brian M. Hoffman, José M. Argüello, Amy C. Rosenzweig

Biochemistry and Molecular Biology Faculty Publications

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine rich N-terminal domain of 130 amino acids in addition to a C-terminal copper-binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and X-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in ...


Human Frataxin: Iron And Ferrochelatase Binding Surface, Krisztina Z. Bencze, Taejin Yoon, CéSar MilláN-Pacheco, Patrick B. Bradley, Nina Pastor, J. A. Cowan, Timothy L. Stemmler May 2007

Human Frataxin: Iron And Ferrochelatase Binding Surface, Krisztina Z. Bencze, Taejin Yoon, CéSar MilláN-Pacheco, Patrick B. Bradley, Nina Pastor, J. A. Cowan, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

The coordinated iron structure and ferrochelatase binding surface of human frataxin have been characterized to provide insight into the protein’s ability to serve as the iron chaperone during heme biosynthesis.


The Structure And Function Of Frataxin, Krisztina Z. Bencze, Kalyan C. Kondapalli, Jeremy D. Cook, Stephen Mcmahon, César Millán-Pacheco, Nina Pastor, Timothy L. Stemmler Oct 2006

The Structure And Function Of Frataxin, Krisztina Z. Bencze, Kalyan C. Kondapalli, Jeremy D. Cook, Stephen Mcmahon, César Millán-Pacheco, Nina Pastor, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich’s ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin’s specific function remains a point of controversy, a general consensus is the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein’s role in ...


Three-Dimensional Structure Of The Bacterial Cell Wall Peptidoglycan, Samy O. Meroueh, Krisztina Z. Bencze, Dusan Hesek, Mijoon Lee, Timothy L. Stemmler, Shahriar Mobashery Mar 2006

Three-Dimensional Structure Of The Bacterial Cell Wall Peptidoglycan, Samy O. Meroueh, Krisztina Z. Bencze, Dusan Hesek, Mijoon Lee, Timothy L. Stemmler, Shahriar Mobashery

Biochemistry and Molecular Biology Faculty Publications

The 3D structure of the bacterial peptidoglycan, the major constit- uent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmu- ramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacte- rium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its ...


The Importance Of A Critical Protonation State And The Fate Of The Catalytic Steps In Class A Β-Lactamases And Penicillin-Binding Proteins, Dasantila Golemi-Kotra, Samy O. Meroueh, Choonkeun Kim, Sergei B. Vakulenko, Alexey Bulychev, Ann J. Stemmler, Timothy L. Stemmler, Shahriar Mobashery May 2004

The Importance Of A Critical Protonation State And The Fate Of The Catalytic Steps In Class A Β-Lactamases And Penicillin-Binding Proteins, Dasantila Golemi-Kotra, Samy O. Meroueh, Choonkeun Kim, Sergei B. Vakulenko, Alexey Bulychev, Ann J. Stemmler, Timothy L. Stemmler, Shahriar Mobashery

Biochemistry and Molecular Biology Faculty Publications

b-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to b-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active-site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins and crystal structures demonstrate that the side chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys73) in the ...


Purified Particulate Methane Monooxygenase From Methylococcus Capsulatus (Bath) Is A Dimer With Both Mononuclear Copper And A Copper-Containing Cluster, Raquel L. Lieberman, Deepak B. Shrestha, Peter E. Doan, Brian M. Hoffman, Timothy L. Stemmler, Amy C. Rosenzweig Mar 2003

Purified Particulate Methane Monooxygenase From Methylococcus Capsulatus (Bath) Is A Dimer With Both Mononuclear Copper And A Copper-Containing Cluster, Raquel L. Lieberman, Deepak B. Shrestha, Peter E. Doan, Brian M. Hoffman, Timothy L. Stemmler, Amy C. Rosenzweig

Biochemistry and Molecular Biology Faculty Publications

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of ~200 kDa, probably corresponding to an a2b2g2 polypeptide arrangement. Each 200 kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ...