Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.Fil: Llarrull, Leticia Irene. Universidad Nacional de Rosario; Argentina. University of Notre Dame; Estados Unidos. Universidad Nacional de Rosario; ArgentinaFil: Mobashery, Shahriar. University of Notre Dame-Indiana; Estados Unido

Roles of Matrix Metalloproteinases in Cutaneous Wound Healing

Wound healing is a complex process that consists of hemostasis and inflammation, angiogenesis, re-epithelialization, and tissue remodeling. Matrix metalloproteinases (MMPs) play important roles in wound healing, and their dysregulation leads to prolonged inflammation and delayed wound healing. There are 24 MMPs in humans, and each MMP exists in three forms, of which only the active MMPs play a role in the pathology or repair of wounds. The current methodology does not distinguish between the three forms of MMPs, making it challenging to investigate the roles of MMPs in pathology and wound repair. We used a novel MMP-inhibitor-tethered affinity resin that binds only the active form of MMPs, from which we identified and quantified active MMP-8 and active MMP-9 in a murine diabetic model with delayed wound healing. We showed that up-regulation of active MMP-9 plays a detrimental role whereas active MMP-8 is involved in repairing the wound in diabetic mice. These studies identified MMP-9 as a novel target for therapeutic intervention in the treatment of chronic wounds. A selective inhibitor of MMP-9 that leaves MMP-8 unaffected would provide the most effective therapy and represents a promising strategy for therapeutic intervention in the treatment of diabetic foot ulcers

The X-ray structure of carboxypeptidase a inhibited by a thiirane mechanism-based ihibitor

The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 Å resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1' specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.Fil: Fernández, Daniel. Universitat Autònoma de Barcelona; EspañaFil: Testero, Sebastian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. University of Notre Dame; Estados UnidosFil: Vendrell, Josep. Universitat Autònoma de Barcelona; EspañaFil: Avilés, Francesc X.. Universitat Autònoma de Barcelona; EspañaFil: Mobashery, Shahriar. University of Notre Dame; Estados Unido

Exploring the functional space of thiiranes as gelatinase inhibitors using click chemistry

A series of 4-[(triazolyl)methoxy]phenyl analogs of the phenoxyphenyl-substituted thiirane SB-3CT 1 was evaluated for its ability to inhibit gelatinases, members of the matrix metalloproteinase family of enzymes. The triazole segment of these inhibitors was assembled using the Meldal-Sharpless copper-catalyzed Huisgen dipolar cycloaddition of an azide and a terminal alkyne. While these triazole derivatives possessed fair activity as gelatinase inhibitors, an intermediate used in the dipolar cycloaddition, 4-(propargyloxy)phenyl derivative 2, showed very good activity (<50% inhibitory activity following a 3 h pre-incubation of 2 at a concentration of 3 μM) as an inhibitor of human matrix metalloproteinase-2.Fil: Testero, Sebastian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Fisher, Jed F.. University of Notre Dame; Estados UnidosFil: Chang, Mayland. University of Notre Dame; Estados UnidosFil: Mobashery, Shahriar. University of Notre Dame; Estados Unido

Matrix metalloproteinase proteolysis after stroke : a surrogate indicator for early diagnosis and validation of treatment

Matrix metalloproteinases (MMPs) are a family of endoproteaseses that have various functions from development to disease. They are also believed to play critical roles in the central nerve system for the pathogenesis of stroke. In ischemic stroke, MMP 9 is involved in neuronal apoptosis, edema, and hemorrhagic transformation. In stroke models, MMPs degrade ECM components and disrupt neurovascular integrity, resulting in blood-brain barrier (BBB) disruption and hemorrhage which further damage to the ischemic area. There are experimental pharmacological treatments with MMP inhibitors that decrease the extent of neuronal apoptosis and hemorrhage. Recently we tested a new class of mechanism-based MMP-9 specific inhibitors SB-3CT. Our hypothesis is that MMP-9 causes proteolytic changes resulting in neurovascular damage after focal cerebral ischemia in mice. Inhibition of MMP proteolysis with SB-3CT, should result in decreased apoptosis of neurons and improved behavioral outcomes."Supported in part by the grants of NIH/NIEHS&NCCAM, Dana Foundation, AHA to Z.G.

Recognition of peptidoglycan and b-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics

The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

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 TEM-1 b-lactamase from Escherichia coli. These techniques included perturbation of the pKa of Lys73 by the study of the g-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance and by computational treatment using the thermodynamic integration method. All three methods indicated that the pKa of Lys73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu166 and Lys73 of class A b-lactamases has actually raised the pKa of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot definitively rule out that Glu166 activates the active site water, which in turn promotes Ser70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu166 and unprotonated Lys73. As such, unprotonated Lys73 could promote serine for acylation, a process that should be shared among all active-site-serine b-lactamases and penicillin-binding proteins

Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC

7 pags, 3 figs, 1 tabThe first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division. © 2010 Nature America, Inc. All rights reserved.This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711, SAF2006-00390), EU-CP223111 (CAREPNEUMO, European Union), the COMBACT program (S-BIO-0260/2006) and CIBER de Enfermedades Respiratorias (CIBERES). CIBERES is an initiative of Instituto de Salud Carlos III. The work in the US was supported by the US National Institutes of Health. W.V. was supported by the European Commission (EUR-INTAFAR project). I.P.-D. was a fellow of the Consejo Superior de Investigaciones Científicas

Mechanism-based Inhibitor of Matrix Metalloproteinase-9 Rescues Brain from Focal Cerebral Ischemia-induced Damage and Improve Neurological Outcomes in Mice [abstract]

Neuroscience - Vision & Functional Brain Imaging Poster SessionStroke is the third leading cause of death in the US and the primary cause of long-term disability. Acute ischemic stroke, the most common form of stroke, is caused by clotting in the cerebral arteries leading to brain oxygen deprivation and cerebral infarction. The events involved in stroke include brain cell injury or death, breakdown of the blood-brain barrier (BBB), edema, and hemorrhage, which are associated with the expression and activation of matrix metalloproteinases (MMPs), particularly MMP-9. In two focal cerebral ischemia paradigms - the filament-induced transient middle cerebral artery occlusion (MCAo) and the embolus-induced permanent MCAo in mice, we examined MMP-9 proteolysis of extracellular matrix (ECM) components and the neuroprotective effects of the highly selective mechanism-based inhibitor of MMP-9, SB-3CT, which is activated by MMP-9 under pathological conditions. We demonstrated that MMP-9 degrades the ECM protein laminin and that this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice. SB-3CT dramatically blocks MMP-9 activity and decreases MMP-9-mediated laminin cleavage, thus rescuing neurons from apoptosis and ameliorating neurobehavioral outcomes. Significant therapeutic activity of SB-3CT is seen up to 6 h after initial brain damage. Moreover, treatment with SB-3CT attenuates brain MMP-9 activity and protects against delayed neuronal cell death in the embolus-induced permanent MCAo in mice. We conclude that MMP-9 is a highly promising drug target and that SB-3CT has significant therapeutic potential in stroke patients

La importancia de los intereses académicos en la política científica y tecnológica catalana

Publicado en: 'Papers: Revista de Sociología', 70: 11-40, 2003Este artículo describe la emergencia y orientación de las políticas de I+D e innovación en Cataluña. Se analizan cuáles son los factores más influyentes en la orientación de estas políticas y, en definitiva, en las opciones políticas que se toman. La política de ciencia y tecnología desarrollada por el gobierno regional catalán desde principios de los años ochenta ha sido una política en la que, a pesar de las preferencias manifestadas en el discurso político, ha predominado un modelo de política académico sobre el de orientación empresarial. Asimismo, en términos organizativos e institucionales, en la Administración autonómica, la política científica ha estado separada y diferenciada de la política tecnológica a pesar del diseño inicial de instituciones interdepartamentales. La principal razón de que la política de I+D catalana no siguiera un modelo más industrial, ligado al mundo empresarial, fue la presión que ejercieron las universidades catalanas para que, tanto el diseño institucional como el contenido de la política se adaptara a sus necesidades. La trayectoria académica previa de los gestores también contribuyó a la reorientación de las preferencias políticas. A pesar de la importancia de las empresas catalanas en la I+D, éstas no se movilizaron ni presionaron a los gobiernos suficientemente. Analíticamente, este caso ilustra cómo la sola creación política de instituciones no garantiza la realización de las preferencias políticas. También pone de manifiesto cómo el horizonte temporal de la toma de decisiones gubernamental tiene un efecto en las expectativas de los actores, que desarrollan procesos de aprendizaje a partir de las experiencias en arenas políticas similares a otros niveles. Por último, destaca la importancia del poder en las instituciones de gestión en este tipo de política distributiva.Peer reviewe