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Dive into the research topics where Gabriel Rosenblum is active.

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Featured researches published by Gabriel Rosenblum.


Biochimica et Biophysica Acta | 2010

Structural and functional bases for allosteric control of MMP activities: can it pave the path for selective inhibition?

Netta Sela-Passwell; Gabriel Rosenblum; Tsipi Shoham; Irit Sagi

The zinc-dependent matrix metalloproteinases (MMPs) belong to a large family of structurally homologous enzymes. These enzymes are involved in a wide variety of biological processes ranging from physiological cell proliferation and differentiation to pathological states associated with tumor metastasis, inflammation, tissue degeneration, and cell death. Controlling the enzymatic activity of specific individual MMPs by antagonist molecules is highly desirable, first, for studying their individual roles, and second as potential therapeutic agents. However, blocking the enzymatic activity with synthetic small inhibitors appears to be an extremely difficult task. Thus, this is an unmet need presumably due to the high structural homology between MMP catalytic domains. Recent reports have recognized a potential role for exosite or allosteric protein regions, distinct from the extended catalytic pocket, in mediating MMP activation and substrate hydrolysis. This raises the possibility that MMP enzymatic and non-enzymatic activities may be modified via antagonist molecules targeted to such allosteric sites or to alternative enzyme domains. In this review, we discuss the structural and functional bases for potential allosteric control of MMPs and highlight potential alternative enzyme domains as targets for designing highly selective MMP inhibitors.


Molecular Cell | 2011

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

Chunlai Chen; Benjamin H. Stevens; Jaskarin Kaur; Diana Cabral; Hanqing Liu; Yuhong Wang; Haibo Zhang; Gabriel Rosenblum; Zeev Smilansky; Yale E. Goldman; Barry S. Cooperman

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.


PLOS ONE | 2010

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Gabriel Rosenblum; Philippe E. Van den Steen; Sidney R. Cohen; Arkady Bitler; David D. Brand; Ghislain Opdenakker; Irit Sagi

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.


FEBS Letters | 2014

Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses

Gabriel Rosenblum; Barry S. Cooperman

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over‐expressed protein. The flexible nature of such cell‐free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.


Journal of the American Chemical Society | 2013

QUANTIFYING ELONGATION RHYTHM DURING FULL-LENGTH PROTEIN SYNTHESIS

Gabriel Rosenblum; Chunlai Chen; Jaskiran Kaur; Xiaonan Cui; Haibo Zhang; Haruichi Asahara; Shaorong Chong; Zeev Smilansky; Yale E. Goldman; Barry S. Cooperman

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.


Nucleic Acids Research | 2012

Real-time assay for testing components of protein synthesis

Gabriel Rosenblum; Chunlai Chen; Jaskiran Kaur; Xiaonan Cui; Yale E. Goldman; Barry S. Cooperman

We present a flexible, real-time-coupled transcription–translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.


Archive | 2011

Mechanism and dynamics of the elongation cycle

Barry S. Cooperman; Yale E. Goldman; Chunlai Chen; Ian Farrell; Jaskarin Kaur; Hanqing Liu; Wei Liu; Gabriel Rosenblum; Zeev Smilansky; Benjamin H. Stevens; Haibo Zhang

Continued dramatic progress in the elucidation of the structures of the bacterial ribosome and its functional complexes has led to proposals for the detailed mechanisms of ribosome-catalyzed protein synthesis (Schmeing and Ramakrishnan, 2009; Agirrezabala and Frank, 2009). Ensemble rapid reaction kinetics (Antoun et al., 2006; Daviter et al., 2006; Dorner et al., 2006; Grigoriadou et al., 2007; Hetricket al., 2009; Pan et al., 2007, 2008; Pape et al., 1998; Phelps and Joseph 2006; Rodnina et al., 1997; Savelsbergh et al., 2003; Walker et al., 2008; Wintermeyer et al., 2004; Zaher and Green, 2009; Zavialov and Ehrenberg, 2003) and single-molecule (Blanchard et al., 2004a, b; Cornish et al., 2008, 2009; Fei et al., 2008, 2009; Marshall et al., 2008, 2009; Munro et al., 2007, 2010a, b; Uemura et al., 2010; Wang et al., 2007) studies of the translational machinery in the past several years have resulted in increased understanding of many aspects of the initiation, elongation, and termination phases of protein synthesis, but many essential points remain to be elucidated.


Proceedings of the National Academy of Sciences of the United States of America | 2018

Slow domain reconfiguration causes power-law kinetics in a two-state enzyme

Iris Grossman-Haham; Gabriel Rosenblum; Trishool Namani; Hagen Hofmann

Significance Dynamic disorder in enzyme catalysis due to conformational heterogeneity is widespread in nature. However, the structural origin for such conformational multiplicity is often elusive. Our results show that the opening and closing of two domains in the redox enzyme QSOX is hampered by a broad ensemble of slowly exchanging open conformations. This heterogeneity is a direct result of the disordered interdomain linker paired with interactions between the domains. Since the effective domain concentration in natural fusions of distinct modules such as in QSOX is very high, interdomain interactions can be persistent, thus resulting in slow sampling. We therefore expect that multidomain enzymes are particularly prone to catalytic disorder such as memory effects. Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.


Bioconjugate Chemistry | 2013

Tb3+-tRNA for LRET studies of Protein Synthesis

Dulce Alonso; Wei Liu; Gabriel Rosenblum; Tomoyasu Mani; Yale E. Goldman; Barry S. Cooperman

When suitably labeled bulk tRNAs are transfected into cells they give rise to FRET (fluorescence resonance energy transfer) signals via binding to ribosomes that provide a measure of total protein synthesis. Application of this approach to monitoring rates of specific protein synthesis requires achieving a very high signal-to-noise ratio. Such high ratios may be attainable using LRET (luminescence resonance energy transfer) in place of FRET. Lanthanide complexes containing an antenna chromophore are excellent LRET donors. Here we describe the synthesis of a Phe-tRNA(Phe) labeled with a Tb(3+) complex, denoted Tb(3+)-Phe-tRNA(Phe) that, notwithstanding the bulkiness of the Tb(3+) complex, is active in protein synthesis.


Structure | 2007

Insights into the Structure and Domain Flexibility of Full-Length Pro-Matrix Metalloproteinase-9/Gelatinase B

Gabriel Rosenblum; Philippe E. Van den Steen; Sidney R. Cohen; J. Günter Grossmann; Jessica Frenkel; Rotem Sertchook; Nelle Slack; Richard W. Strange; Ghislain Opdenakker; Irit Sagi

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Irit Sagi

Weizmann Institute of Science

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Yale E. Goldman

University of Pennsylvania

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Chunlai Chen

University of Pennsylvania

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Haibo Zhang

University of Pennsylvania

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Zeev Smilansky

University of Pennsylvania

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Jaskiran Kaur

University of Pennsylvania

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Xiaonan Cui

University of Pennsylvania

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Sidney R. Cohen

Weizmann Institute of Science

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