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1.
J Biol Chem ; 295(27): 8945-8957, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32371399

RESUMO

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)- and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

3.
Commun Biol ; 2: 345, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31552298

RESUMO

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

4.
Cell Rep ; 26(11): 2916-2928.e13, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30865883

RESUMO

The chromatin-associated protein WDR5 is a promising target for pharmacological inhibition in cancer. Drug discovery efforts center on the blockade of the "WIN site" of WDR5, a well-defined pocket that is amenable to small molecule inhibition. Various cancer contexts have been proposed to be targets for WIN site inhibitors, but a lack of understanding of WDR5 target genes and of the primary effects of WIN site inhibitors hampers their utility. Here, by the discovery of potent WIN site inhibitors, we demonstrate that the WIN site links WDR5 to chromatin at a small cohort of loci, including a specific subset of ribosome protein genes. WIN site inhibitors rapidly displace WDR5 from chromatin and decrease the expression of associated genes, causing translational inhibition, nucleolar stress, and p53 induction. Our studies define a mode by which WDR5 engages chromatin and forecast that WIN site blockade could have utility against multiple cancer types.


Assuntos
Cromatina/metabolismo , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Masculino , Ligação Proteica/efeitos dos fármacos
5.
Protein Sci ; 28(4): 808-822, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30663186

RESUMO

The use of Seleno-methionine (SeMet) incorporated protein crystals for single or multi-wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared with native protein expression.


Assuntos
Sobrevivência Celular , Insetos/citologia , Proteínas Recombinantes/metabolismo , Selenometionina/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Expressão Gênica , Insetos/genética , Insetos/metabolismo , Modelos Moleculares , Proteínas Recombinantes/genética
6.
J Hum Hypertens ; 32(11): 770-774, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30232401

RESUMO

The present study examines the performance of the ambulatory blood pressure monitor PHYSIO-PORT UP in pregnant and preeclamptic women. The systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of 33 subjects (among them 13 with preeclampsia) were sequentially measured and compared to reference measurements. Those reference measurements were taken by two observers using a mercury sphygmomanometer. Subject recruitment and evaluation of the measurement results were performed according to the European Society of Hypertension International Protocol revision 2010 (ESH-IP 2010). Three readings were recorded per subject under static conditions. The protocol did not include testing under ambulatory conditions. 87/90 (SBP/DBP) of the 99 readings had a difference ≤ 5 mmHg, 96/98 had a difference 98 ≤ 10 mmHg, and 98/99 had a difference ≤ 15 mmHg. 2 out of 3 readings ≤ 5 mmHg was achieved by 31/32 subjects. None of the subjects had less than 1 comparative reading ≤ 5 mmHg. In conclusion, the PHYSIO-PORT UP fulfilled all requirements of the ESH-IP 2010. According to these results, the PHYSIO-PORT UP can be recommended for blood pressure measurement in pregnant women.


Assuntos
Monitorização Ambulatorial da Pressão Arterial/instrumentação , Feminino , Humanos , Variações Dependentes do Observador , Gravidez
7.
PLoS One ; 13(4): e0195356, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29614134

RESUMO

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.


Assuntos
Baculoviridae/genética , Vetores Genéticos , Complexos Multiproteicos/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/metabolismo , Western Blotting , Contagem de Células , Citometria de Fluxo , Expressão Gênica , Modelos Teóricos , Complexos Multiproteicos/genética , Controle de Qualidade , Proteínas Recombinantes/genética , Células Sf9 , Solubilidade
8.
Ultramicroscopy ; 182: 233-242, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28734230

RESUMO

A combined X-ray and scanning tunneling microscopy (STM) instrument is presented that enables the local detection of X-ray absorption on surfaces in a gas environment. To suppress the collection of ion currents generated in the gas phase, coaxially shielded STM tips were used. The conductive outer shield of the coaxial tips can be biased to deflect ions away from the tip core. When tunneling, the X-ray-induced current is separated from the regular, 'topographic' tunneling current using a novel high-speed separation scheme. We demonstrate the capabilities of the instrument by measuring the local X-ray-induced current on Au(1 1 1) in 800 mbar Ar.

9.
Sci Rep ; 6: 28419, 2016 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-27341298

RESUMO

Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.


Assuntos
Proteínas Priônicas/química , Xenônio/química , Sequência de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Terciária de Proteína , Marcadores de Spin
10.
Nucleic Acids Res ; 42(17): 10975-86, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25183520

RESUMO

Cdc48/p97 is an evolutionary conserved ubiquitin-dependent chaperone involved in a broad array of cellular functions due to its ability to associate with multiple cofactors. Aside from its role in removing RNA polymerase II from chromatin after DNA damage, little is known about how this AAA-ATPase is involved in the transcriptional process. Here, we show that yeast Cdc48 is recruited to chromatin in a transcription-coupled manner and modulates gene expression. Cdc48, together with its cofactor Ubx3 controls monoubiquitylation of histone H2B, a conserved modification regulating nucleosome dynamics and chromatin organization. Mechanistically, Cdc48 facilitates the recruitment of Lge1, a cofactor of the H2B ubiquitin ligase Bre1. The function of Cdc48 in controlling H2B ubiquitylation appears conserved in human cells because disease-related mutations or chemical inhibition of p97 function affected the amount of ubiquitylated H2B in muscle cells. Together, these results suggest a prominent role of Cdc48/p97 in the coordination of chromatin remodeling with gene transcription to define cellular differentiation processes.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética , Ubiquitinação , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Mutação , Mioblastos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Proteína com Valosina
11.
Annu Rev Biochem ; 81: 177-201, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22404630

RESUMO

Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription-from transcription initiation through to export of mRNA from the nucleus-is influenced by the UPS and that all major arms of the system--from the first step in ubiquitin (Ub) conjugation through to the proteasome-are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Transcrição Genética , Ubiquitinação , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Fatores de Transcrição/química
12.
Biochem J ; 425(2): 373-80, 2009 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-19860741

RESUMO

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176-273) at a resolution of 1.55 A (1 A=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE" appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.


Assuntos
Proteínas Nucleares/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Fatores de Elongação da Transcrição/química , Cristalografia por Raios X , Glutamina , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Multimerização Proteica , Homologia Estrutural de Proteína
13.
Biochem Biophys Res Commun ; 370(3): 414-8, 2008 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-18373978

RESUMO

The eukaryotic transcription elongation factor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF), is involved in regulating the processivity of RNA polymerase II. DSIF plays also a role in transcriptional activation, and in concert with the negative elongation factor NELF causes promoter proximal pausing of RNA polymerase II. Furthermore, DSIF has also been implicated in regulating the transcription of the human immunodeficiency virus proviral DNA. Human DSIF is composed of the two subunits, hSpt4 (p14) and hSpt5 (p160), corresponding to the yeast homologs Spt4 and Spt5. Here we show the purification and characterization of the small subunit, hSpt4. We were able to purify the protein in a soluble form separately from the larger hSpt5 subunit. CD and NMR spectroscopy show that the purified protein hSpt4 exhibits an alpha/beta topology with a well defined tertiary structure. Furthermore metal analysis by ICP-OES indicates that the protein contains a functional 4-Cys Zn-finger.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Proteínas Repressoras/química , Proteínas Repressoras/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Dedos de Zinco , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/isolamento & purificação , Zinco/análise , Zinco/metabolismo
14.
Biochemistry ; 47(12): 3756-61, 2008 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-18303858

RESUMO

The E subunit of the human heterotetrameric negative transcription elongation factor (NELF-E) contains a canonical betaalphabetabetaalphabeta RNA recognition motif (RRM) that binds to a wide variety of RNA sequences. These induce very similar conformational changes in the RRM as determined by nuclear magnetic resonance spectroscopy. Although the RNA binding interface of a canonical RRM is mainly located at its beta-sheet surface, for NELF-E RRM large chemical shift perturbations are observed for residues in the flexible C-terminal region and the loop between beta 3 and alpha 2, and both regions are distant from the interface. We determined the solution structure of single-stranded transactivator responsive element (TAR) RNA-bound NELF-E RRM. This structure clearly shows that RNA binding to NELF-E RRM induces formation of a helix in the C-terminus. The RNA-bound form of NELF-E RRM is very similar to the RNA-bound form of U1A RRM, although the C-terminus of the NELF-E RRM is unstructured in the free protein, whereas it is helical in the U1A protein. Thus, RNA binding to NELF-E RRM induces a conformational change toward the U1A structure, resulting in highly similar RNA binding conformations for both proteins.


Assuntos
Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Fatores de Elongação da Transcrição/química , Motivos de Aminoácidos , Sequência de Bases , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ribonucleoproteína Nuclear Pequena U1/química , Fatores de Transcrição
15.
Biochem J ; 400(3): 449-56, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16898873

RESUMO

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.


Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Fatores de Transcrição
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