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1.
PLoS Pathog ; 13(7): e1006515, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28753640

RESUMEN

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Proteínas de Unión a Fosfato , Fosforilación , Unión Proteica , Dominios Proteicos , Técnicas del Sistema de Dos Híbridos
2.
RNA ; 20(4): 447-61, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24570482

RESUMEN

RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.


Asunto(s)
Aptámeros de Nucleótidos/farmacología , Muramidasa/antagonistas & inhibidores , Muramidasa/metabolismo , Sodio/metabolismo , Animales , Aptámeros de Nucleótidos/química , Emparejamiento Base , Secuencia de Bases , Catálisis , Pollos , Espectroscopía de Resonancia Magnética , Micrococcus/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/genética , Conformación de Ácido Nucleico , Concentración Osmolar , Unión Proteica , Conformación Proteica , Sodio/química , Electricidad Estática , Especificidad por Sustrato , Ultracentrifugación , Difracción de Rayos X
3.
J Biol Chem ; 289(37): 25750-63, 2014 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-25074926

RESUMEN

TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.


Asunto(s)
Apoptosis/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Sanguíneas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Jurkat , Proteína S , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Estomatitis Vesicular/genética , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor Axl
4.
J Biol Chem ; 289(37): 25737-49, 2014 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-25074939

RESUMEN

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.


Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Apoptosis/genética , Neoplasias de la Mama/patología , Movimiento Celular/genética , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Fagocitosis/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor Axl
5.
PLoS Pathog ; 5(5): e1000460, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19478878

RESUMEN

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.


Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/etiología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Enfermedad Crónica , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Cobayas , Ratones , Proteínas de Unión a Fosfato , Unión Proteica , Tuberculosis/patología
6.
J Mol Biol ; 342(5): 1559-67, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15364581

RESUMEN

Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.


Asunto(s)
Actinas/química , Actinas/metabolismo , Proteínas de Microfilamentos/farmacología , Conformación Proteica/efectos de los fármacos , Factores Despolimerizantes de la Actina , Reactivos de Enlaces Cruzados , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Subtilisina/metabolismo , Tripsina/metabolismo
7.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 61(Pt 11): 959-63, 2005 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-16511207

RESUMEN

The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7 A resolution. YbeY is a hypothetical protein that belongs to the UPF0054 family. The structure reveals that the protein binds a metal ion in a tetrahedral geometry. Three coordination sites are provided by histidine residues, while the fourth might be a water molecule that is not seen in the diffraction map because of its relatively low resolution. X-ray fluorescence analysis of the purified protein suggests that the metal is a nickel ion. The structure of ybeY and its sequence similarity to a number of predicted metal-dependent hydrolases provides a functional assignment for this protein family. The figures and tables of this paper were prepared using semi-automated tools, termed the Autopublish server, developed by the New York Structural GenomiX Research Consortium, with the goal of facilitating the rapid publication of crystallographic structures that emanate from worldwide Structural Genomics efforts, including the NIH-funded Protein Structure Initiative.


Asunto(s)
Proteínas de Escherichia coli/química , Metaloproteínas/química , Secuencia de Aminoácidos , Clonación Molecular , Biología Computacional , Cristalografía por Rayos X , Proteínas de Escherichia coli/fisiología , Histidina/química , Metaloproteínas/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia/métodos , Difracción de Rayos X
8.
Chem Biol ; 19(6): 721-30, 2012 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-22726686

RESUMEN

Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here, we present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Organofosfonatos/farmacología , Pentosiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Profármacos/farmacología , Dominio Catalítico/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Eritrocitos/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Organofosfonatos/síntesis química , Organofosfonatos/química , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Profármacos/síntesis química , Profármacos/química , Relación Estructura-Actividad
9.
Biochem Biophys Res Commun ; 307(4): 928-34, 2003 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-12878200

RESUMEN

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteómica/métodos , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Péptidos/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
10.
J Biol Chem ; 279(18): 18103-6, 2004 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-14982926

RESUMEN

Purine nucleoside phosphorylase from Plasmodium falciparum (PfPNP) is an anti-malarial target based on the activity of Immucillins. The crystal structure of PfPNP.Immucillin-H (ImmH).SO(4) reveals a homohexamer with ImmH and SO(4) bound at each catalytic site. A solvent-filled cavity close to the 5'-hydroxyl group of ImmH suggested that PfPNP can accept additional functional groups at the 5'-carbon. Assays established 5'-methylthioinosine (MTI) as a substrate for PfPNP. MTI is not found in human metabolism. These properties of PfPNP suggest unusual purine pathways in P. falciparum and provide structural and mechanistic foundations for the design of malaria-specific transition state analogue inhibitors. 5'-Methylthio-Immucillin-H (MT-ImmH) was designed to resemble the transition state of PfPNP and binds to PfPNP and human-PNP with K(d) values of 2.7 and 303 nm, respectively, to give a discrimination factor of 112. MT-ImmH is the first inhibitor that favors PfPNP inhibition. The structure of PfPNP.MT-ImmH.SO(4) shows that the hydrophobic methylthio group inserts into a hydrophobic region adjacent to the more hydrophilic 5'-hydroxyl binding site of ImmH. The catalytic features of PfPNP indicate a dual cellular function in purine salvage and polyamine metabolism. Combined metabolic functions in a single enzyme strengthen the rationale for targeting PfPNP in anti-malarial action.


Asunto(s)
Metiltioinosina/análogos & derivados , Plasmodium falciparum/enzimología , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Animales , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metiltioinosina/metabolismo , Estructura Molecular , Nucleósidos de Purina , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinonas/química , Pirroles/química
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