Biomedical subjects

Alex Y Strongin

Publications and source records attributed to Alex Y Strongin.

At least 19 recordsLinked to original sources

PubMed2007

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Binding Sites↗

PubMed2006

Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B-NS3 junction region.

Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.

Amino Acid Motifs↗

PubMed2006

Membrane type-1 matrix metalloproteinase confers aneuploidy and tumorigenicity on mammary epithelial cells.

An elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with multiple malignancies. Recently, we discovered that recycled MT1-MMP was trafficked along the tubulin cytoskeleton into the centrosomal compartment and cleaved the integral centrosomal protein pericentrin-2. These events correlated with the induction of chromosome instability and aneuploidy in nonmalignant Madine-Darby canine kidney cells. Accordingly, we hypothesized that MT1-MMP is an oncogene that promotes malignant transformation of normal cells rather than just an enzyme that supports growth of preexisting tumors. To prove our hypothesis, we transfected normal 184B5 human mammary epithelial cells with MT1-MMP (184B5-MT1 cells). MT1-MMP was colocalized with pericentrin in the centrosomal compartment and especially in the midbody of dividing cells. 184B5-MT1 cells acquired the ability to activate MMP-2, to cleave pericentrin, and to invade the Matrigel matrix. 184B5-MT1 cells exhibited aneuploidy, and they were efficient in generating tumors in the orthotopic xenograft model in immunodeficient mice. Because of the absence of tumor angiogenesis and the resulting insufficient blood supply, the tumors then regressed with significant accompanying necrosis. Gene array studies confirmed a significant up-regulation of oncogenes and tumorigenic genes but not the angiogenesis-promoting genes in 184B5-MT1 cells. We believe that our data point to a novel function of MT1-MMP in the initial stages of malignant transformation and to new and hitherto unknown transition mechanism from normalcy to malignancy.

Aneuploidy↗

PubMed2006

Interference with the complement system by tumor cell membrane type-1 matrix metalloproteinase plays a significant role in promoting metastasis in mice.

Neoplasms have developed strategies to protect themselves against the complement-mediated host immunity. Invasion- and metastasis-promoting membrane type-1 (MT1) matrix metalloproteinase (MMP) is strongly associated with many metastatic cancer types. The relative importance of the individual functions of MT1-MMP in metastasis was, however, unknown. We have now determined that the expression of murine MT1-MMP in murine melanoma B16F1 cells strongly increased the number of metastatic loci in the lungs of syngeneic C57BL/6 mice. In contrast, MT1-MMP did not affect the number of metastatic loci in complement-deficient C57BL/6-C3-/- mice. Our results indicated, for the first time, that the anticomplement activity of MT1-MMP played a significant role in promoting metastasis in vivo and determined the relative importance of the anticomplement activity in the total metastatic effect of this multifunctional proteolytic enzyme. We believe that our results shed additional light on the functions of MT1-MMP in cancer and clearly make this protease a promising drug target in metastatic malignancies.

Animals↗

PubMed2006

O-glycosylation regulates autolysis of cellular membrane type-1 matrix metalloproteinase (MT1-MMP).

MT1-MMP is a key enzyme in cancer cell invasion and metastasis. The activity of cellular MT1-MMP is regulated by furin-like proprotein convertases, TIMPs, shedding, autoproteolysis, dimerization, exocytosis, endocytosis, and recycling. Our data demonstrate that, in addition to these already known mechanisms, MT1-MMP is regulated by O-glycosylation of its hinge region. Insignificant autolytic degradation is characteristic for naturally expressed, glycosylated, MT1-MMP. In turn, extensive autolytic degradation, which leads to the inactivation of the protease and the generation of its C-terminal membrane-tethered degraded species, is a feature of overexpressed MT1-MMP. We have determined that incomplete glycosylation stimulates extensive autocatalytic degradation and self-inactivation of MT1-MMP. Self-proteolysis commences during the secretory process of MT1-MMP through the cell compartment to the plasma membrane. The strongly negatively charged sialic acid is the most important functional moiety of the glycopart of MT1-MMP. We hypothesize that sialic acid of the O-glycosylation cassette restricts the access of the catalytic domain to the hinge region and to the autolytic cleavage site and protects MT1-MMP from autolysis. Overall, our results point out that there is a delicate balance between glycosylation and self-proteolysis of MT1-MMP in cancer cells and that when this balance is upset the catalytically potent MT1-MMP pool is self-proteolyzed.

Animals↗

PubMed2006

Matrix metalloproteinase 26 proteolysis of the NH2-terminal domain of the estrogen receptor beta correlates with the survival of breast cancer patients.

Estrogens have many cellular functions, including their interactions with estrogen receptors alpha and beta (ERalpha and ERbeta). Earlier, we determined that the estrogen-ER complex stimulates the transcriptional activity of the matrix metalloproteinase 26 (MMP-26) gene promoter. We then determined that ERbeta is susceptible to MMP-26 proteolysis whereas ERalpha is resistant to the protease. MMP-26 targets the NH(2)-terminal region of ERbeta coding for the divergent NH(2)-terminal A/B domain that is responsible for the ligand-independent transactivation function. As a result, MMP-26 proteolysis generates the COOH-terminal fragments of ERbeta. Immunohistochemical analysis of tissue microarrays derived from 121 cancer patients corroborated these data and revealed an inverse correlation between the ERalpha-dependent expression of MMP-26 and the levels of the intact ERbeta in breast carcinomas. MMP-26 is not expressed in normal mammary epithelium. The levels of MMP-26 are strongly up-regulated in ductal carcinoma in situ (DCIS). In the course of further disease progression through stages I to III, the expression of MMP-26 decreases. In contrast to many tumor-promoting MMPs, the expression of MMP-26 in DCIS correlated with a longer patient survival. Our data suggest the existence of an MMP-26-mediated intracellular pathway that targets ERbeta and that MMP-26, a novel and valuable cancer marker, contributes favorably to the survival of the ERalpha/beta-positive cohort of breast cancer patients.

Breast Neoplasms↗

PubMed2006

Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase.

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.

Amino Acid Sequence↗

PubMed2006

Anthrax lethal factor protease inhibitors: synthesis, SAR, and structure-based 3D QSAR studies.

We have recently identified a series of compounds that efficiently inhibit anthrax lethal factor (LF) metallo-protease. Here we present further structure-activity relationship and CoMFA (comparative molecular field analysis) studies on newly derived inhibitors. The obtained 3D QSAR model was subsequently compared with the X-ray structure of the complex between LF and a representative compound. Our studies form the basis for the rational design of additional compounds with improved activity and selectivity.

Antigens, Bacterial↗

PubMed2006

Mislocalization and unconventional functions of cellular MMPs in cancer.

MMPs are multifunctional enzymes capable of targeting the extracellular matrix, growth factors, cytokines and cell surface-associated adhesion and signaling receptors. The cellular localization and the activity of MMPs are tightly controlled at both the transcriptional and the post-transcriptional levels. Mislocalization and presentation in unconventional cellular compartments provide MMPs with an opportunity to cleave previously unidentified proteins. This review is focused on two, entirely different MMPs, one of which is membrane-tethered and another of which is soluble (MT1-MMP and MMP-26, respectively) from twenty four known human MMPs. Our recent studies determined that both of these enzymes functioned at unexpected cellular compartments and it was resulted in the identification of novel proteolytic pathways, whose significance we only partially comprehend as of this writing. It is reasonable, however, to hypothesize from these data that many individual MMPs perform in a similar manner and display a much broader range of functions compared to what we earlier thought.

Animals↗

PubMed2006

Mechanistic insights into targeting T cell membrane proteinase to promote islet beta-cell rejuvenation in type 1 diabetes.

It has been well established that invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP), a multifunctional membrane-tethered enzyme, functions in cancer cells as a mediator of pericellular proteolysis and directly cleaves several cell surface receptors, including CD44. In this report, we confirm that adhesion of diabetogenic T cells promotes the activation of endogenous MT1-MMP. Activated protease then cleaves CD44 in adherent T cells. We have validated that the T cell CD44 receptor is critical for the adhesion of diabetogenic insulin-specific, CD8-positive, K(d)-restricted cells to the matrix as well as for the subsequent transmigration of the adherent T cells through the endothelium and homing of the transmigrated T cells into the pancreatic islets. We have determined that the inhibition of MT1-MMP by low dosages of AG3340 (a subnanomolar range hydroxamate inhibitor of MMPs that has been widely tested in cancer patients) inhibited both T cell MT1-MMP activity and MT1-MMP-dependent shedding of CD44, immobilized T cells on the endothelium, repressed the homing of diabetogenic T cells into the pancreatic islets, reduced insulitis and mononuclear cell infiltration, and promoted either the recovery or the rejuvenation of the functional insulin-producing beta cells in diabetic NOD mice with freshly developed type I diabetes (IDDM). We believe our data constitute a mechanistic and substantive rationale for clinical trials of selected MT1-MMP inhibitors in the therapy of IDDM in humans.

Animals↗

PubMed2005

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency.

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.

Animals↗

PubMed2005

Centrosomal pericentrin is a direct cleavage target of membrane type-1 matrix metalloproteinase in humans but not in mice: potential implications for tumorigenesis.

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits distinctive and important pericellular cleavage functions. Recently, we determined that MT1-MMP was trafficked to the centrosomes in the course of endocytosis. Our data suggested that the functionally important, integral, centrosomal protein, pericentrin-2, was a cleavage target of MT1-MMP in human and in canine cells and that the sequence of the cleavage sites were ALRRLLG1156 downward arrow L1157FG and ALRRLLS2068 downward arrow L2069FG, respectively. The presence of Asp-948 at the P1 position inactivated the corresponding site (ALRRLLD948-L949FGD) in murine pericentrin. To confirm that MT1-MMP itself cleaves pericentrin directly, rather than indirectly, we analyzed the cleavage of the peptides that span the MT1-MMP cleavage site. In addition, we analyzed glioma U251 cells, which co-expressed MT1-MMP with the wild type murine pericentrin and the D948G mutant. We determined that the D948G mutant that exhibited the cleavage sequence of human pericentrin was sensitive to MT1-MMP, whereas unmodified murine pericentrin was resistant to proteolysis. Taken together, our results confirm that MT1-MMP cleaves pericentrin-2 in humans but not in mice and that mouse models of cancer probably cannot be used to critically examine MT1-MMP functionality.

Animals↗

PubMed2005

The transmembrane domain is essential for the microtubular trafficking of membrane type-1 matrix metalloproteinase (MT1-MMP).

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades the extracellular matrix, initiates the activation pathway of soluble MMPs and regulates the functionality of cell adhesion signaling receptors, thus playing an important role in many cell functions. Intracellular transport mechanisms, currently incompletely understood, regulate the presentation of MT1-MMP at the cell surface. We have focused our efforts on identifying these mechanisms. To understand the transport of MT1-MMP across the cell, we used substitution and deletion mutants, the trafficking of which was examined using antibody uptake and Chariot delivery experiments. Our experiments have demonstrated that the microtubulin cytoskeleton and the centrosomes (the microtubulin cytoskeleton-organizing centers) are essential for the trafficking and the internalization of MT1-MMP. We determined that after reaching the plasma membrane, MT1-MMP is internalized in the Rab-4-positive recycling endosomes and the Rab-11-positive pericentrosomal recycling endosomes. The microtubular trafficking causes the protease to accumulate in the pericentrosomal region of the cell. We believe that the presence of the transmembrane domain is required for the microtubular vesicular trafficking of MT1-MMP because the soluble mutants are not presented at the cell surface and they are not delivered to the centrosomes. The observed transport mechanisms provide a vehicle for the intracellular targets and, accordingly, for an intracellular cleavage function of MT1-MMP in malignant cells, which routinely overexpress this protease.

Animals↗

PubMed2005

A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia.

Neuronal cell death occurs during many neurodegenerative disorders and stroke. The aberrant, excessive activity of matrix metalloproteinases (MMPs), especially MMP-9, contributes directly to neuron apoptosis and brain damage (Rosenberg et al., 1996; Asahi et al., 2001; Gu et al., 2002; Horstmann et al., 2003). We determined that MMP-9 degrades the extracellular matrix protein laminin and that this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice. We also determined that the highly specific thiirane gelatinase inhibitor SB-3CT blocks MMP-9 activity, including MMP-9-mediated laminin cleavage, thus rescuing neurons from apoptosis. We conclude that MMP-9 is a highly promising drug target and that SB-3CT derivatives have significant therapeutic potential in stroke patients.

Animals↗

PubMed2005

Efficient synthetic inhibitors of anthrax lethal factor.

Inhalation anthrax is a deadly disease for which there is currently no effective treatment. Bacillus anthracis lethal factor (LF) metalloproteinase is an integral component of the tripartite anthrax lethal toxin that is essential for the onset and progression of anthrax. We report here on a fragment-based approach that allowed us to develop inhibitors of LF. The small-molecule inhibitors we have designed, synthesized, and tested are highly potent and selective against LF in both in vitro tests and cell-based assays. These inhibitors do not affect the prototype human metalloproteinases that are structurally similar to LF. Initial in vivo evaluation of postexposure efficacy of our inhibitors combined with antibiotic ciprofloxacin against B. anthracis resulted in significant protection. Our data strongly indicate that the scaffold of inhibitors we have identified is the foundation for the development of novel, safe, and effective emergency therapy of postexposure inhalation anthrax.

Animals↗

PubMed2005

Inhibition of membrane type-1 matrix metalloproteinase by cancer drugs interferes with the homing of diabetogenic T cells into the pancreas.

We have discovered that clinically tested inhibitors of matrix metalloproteinases can control the functional activity of T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the onset of disease in a rodent model of type 1 diabetes in non-obese diabetic mice. We determined that MT1-MMP proteolysis of the T cell surface CD44 adhesion receptor affects the homing of T cells into the pancreas. We also determined that both the induction of the intrinsic T cell MT1-MMP activity and the shedding of cellular CD44 follow the adhesion of insulin-specific, CD8-positive, Kd-restricted T cells to the matrix. Conversely, inhibition of these events by AG3340 (a potent hydroxamate inhibitor that was widely used in clinical trials in cancer patents) impedes the transmigration of diabetogenic T cells into the pancreas and protects non-obese diabetic mice from diabetes onset. Overall, our studies have divulged a previously unknown function of MT1-MMP and identified a promising novel drug target in type I diabetes.

Animals↗

PubMed2005

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits an important intracellular cleavage function and causes chromosome instability.

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in non-malignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation.

Animals↗

PubMed2005

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.

Animals↗