Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversal in vivo using chronic administration of irreversible LOX inhibitor b-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl 4 )-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl 4 withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its res-

Histologically, the adventitial layer is thickened with neovessels sprouting off the vasa vasorum networks. In the aorta, T cells and macrophages can accumulate around microvessels, and histiocytes and T cells form granulomatous infiltrates in the media. Vascular smooth muscle cell dropout leads to medial damage and thinning of the muscular layer. The lamina elastic interna, which separates the media from the intima, is fragmented, granting access for mesenchymal cells migrating toward the intima. The intimal layer is expanded, causing luminal stenosis/occlusion with the vascular lumen of arteries often reduced to a pinhole lesion.

Receptor Ser/Thr protein kinases are candidates for sensors that govern developmental changes and disease processes of Mycobacterium tuberculosis (Mtb), but the functions of these kinases are not established. Here, we show that Mtb protein kinase (Pkn) D overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-anti–sigma factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c, but no other sigma factor regulator, in vitro. In contrast, the purified PknB and PknE kinase domains phosphorylated distinct sigma regulators. Rather than modifying a consensus site, PknD phosphorylated Rv0516c in vitro and in vivo on Thr2 in a unique N-terminal extension. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-anti–sigma factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma factor regulator at an unprecedented control site.

Summary The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate >30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how “back-to-back” N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.

Many bacterial species express ‘eukaryotic-like’ Ser/Thr or Tyr protein kinases and phosphatases that are candidate mediators of developmental changes and host/pathogen interactions. The biological functions of these systems are largely unknown. Recent genetic, biochemical and structural studies have begun to establish a framework for understanding the systems for Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis (Mtb). Ser/Thr protein kinases (STPKs) appear to regulate diverse processes including cell division and molecular transport. Proposed protein substrates of the STPKs include putative regulatory proteins, as well as six proteins containing Forkhead-associated domains. Structures of domains of receptor STPKs and all three Mtb Ser/Thr or Tyr phosphatases afford an initial description of the principal modules that mediate bacterial STPK signaling. These studies revealed that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic STPKs. Several structures also support novel mechanisms of regulation, including dimerization of STPKs, metal-ion binding to PstP and substrate mimicry in PtpB.

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.Tight regulation of Ser/Thr protein kinases (STPKs) 2 is essential for signaling in all three kingdoms of life. In keeping with the importance of phospho-signaling pathways, STPKs are subject to multiple mechanisms of allosteric regulation (1). Diverse regulatory surfaces of kinase domains (KDs) can bind additional proteins (as in the cyclin-dependent kinases and cAMP-dependent protein kinase) or other domains of the kinase itself (such as the Src homology 2 and linker domains of Src). These interactions often affect the assembly of the catalytic site by positioning a conserved, substrate-binding element called the C helix. In addition, phosphorylation of a conserved motif called the activation loop often relieves a steric blockade of the active site and promotes assembly of a substrate-binding platform (1). Although many regulatory interactions have been characterized in eukaryotic protein kinases, the mechanisms by which environmental signals regulate prokaryotic STPKs have yet to be elucidated.Prokaryotic STPKs occur in numerous pathogens and organisms with complex developmental pathways (2). Like their eukaryotic homologs, the bacterial STPKs adopt a characteristic two-domain fold with the ATP-binding site located between the N-and C-terminal lobes (3-5). Unexpectedly, the catalytic domain of the receptor STPK PknB from Mycobacterium tuberculosis formed an unstable, backto-back dimer through an interface in the N-terminal lobe (Fig. 1A) (3-5). Residues in the PknB dimer interface were found to be strictly conserved in orthologs from dozens of bacterial genera, supporting the proposal that KD dimerization plays an important functional role (5). Because the dimer interface included contacts near the C terminus of the C helix, it was ...

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745⅐MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.Human matrix metalloproteinase 9 (MMP9), 3 also referred to as gelatinase B, is a member of the MMP family, which are secreted from cells as inactive zymogens. The MMP family consists of ϳ25 members that share a common domain structure. The proenzyme (pro-MMP9) contains an N-terminal propeptide domain, a zinc-containing catalytic domain with an insertion of three fibronectin type II repeats, and a C-terminal hemopexin-like domain, which is connected to the catalytic domain by an O-glycosylated linker (1). The propeptide domain contains a conserved cysteine residue that coordinates the active site zinc, preventing substrate binding and catalysis. Stepwise proteolytic cleavage of the propeptide is required to generate catalytically competent MMP9 (2, 3). A related protease, MMP3, has been shown to activate MMP9 in vitro by cleaving between residues, Glu-59/Met-60 and Arg-106/Phe-107, releasing the prodomain (4, 5). Once active, MMP9 is capable of cleaving numerous substrates (6).In vivo, MMP9 activity is critical in remodeling components of the extracellular matrix, including collagen IV and laminin in the basement membrane (7). In addition to its role in extracellular matrix remodeling, MMP9 regulates other cellular processes, and its expression and secretion are up-regulated in pathological states such as cancer and chronic inflammation (7-13). MMP9 has also been shown to activate latent cytokines and growth factors and alter both trafficking and cell surface protein expression of both myeloid and lymphoid cells (14 -17).Because of its association with disease and correlation with poor prognosis in ulcerative colitis and colorectal cancer patients, MMP9 has long been considered a potential target for therapeutic intervention. Efforts to discover and develop safe and effective drugs selectively targeting MMP9 have been difficult due to the high structural conservation of the protease active site among MMP family members. A clinical stage, broad-spectrum peptidomimetic MMP inhibitor, marimastat, failed to meet efficacy end points and led to musculoskeletal syndrome in some patients (12,18).A...