Human immunodeficiency virus-1 (HIV-1) Vif is essential for viral evasion of host antiviral factor CEM15/APOBEC3G. We report that Vif interacts with cellular proteins Cul5, elongins B and C, and Rbx1 to form an Skp1-cullin-F-box (SCF)-like complex. The ability of Vif to suppress antiviral activity of APOBEC3G was specifically dependent on Cul5-SCF function, allowing Vif to interact with APOBEC3G and induce its ubiquitination and degradation. A Vif mutant that interacted with APOBEC3G but not with Cul5-SCF was functionally inactive. The Cul5-SCF was also required for Vif function in distantly related simian immunodeficiency virus mac. These results indicate that the conserved Cul5-SCF pathway used by Vif is a potential target for antiviral development.

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Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn 2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.-Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. The extracellular matrix (ECM) of cartilage consists of several types of collagens, proteoglycans, and other noncollagenous macromolecules, all of which interact to form a highly specialized connective tissue (1). Early extracellular cartilage matrix degeneration in arthritis is the result of the action of degradative enzymes. As the severity of arthritis progresses, the synthesis and secretion of matrix-degrading enzymes markedly increase (2). The control 1 These authors contributed equally to this work.

Abstract-The migration of vascular smooth muscle cells (VSMCs) plays an essential role during the development of atherosclerosis and restenosis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases in VSMC migration. A recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), is capable of degrading vascular ECM proteins. Here, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. ADAMTS-7 protein accumulated preferentially in neointima of the carotid artery wall after balloon injury. In primary VSMCs, ADAMTS-7 level was enhanced by the proinflammatory cytokine tumor necrosis factor ␣ and growth factor platelet-derived growth factor-BB. ADAMTS-7 overexpression greatly accelerated and small interfering RNA knockdown markedly retarded VSMC migration/invasion in vitro. In addition, luminal delivery of ADAMTS-7 adenovirus to carotid arteries exacerbated intimal thickening nearly sixfold 7 days after injury. Conversely, perivascular administration of ADAMTS-7 small interfering RNA but not scramble small interfering RNA to injured arteries attenuated intimal thickening by 50% at 14 days after injury. Furthermore, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels. Replenishing COMP circumvented the promigratory effect of ADAMTS-7 on VSMCs. Enforced expression of COMP significantly suppressed VSMC migration and neointima formation postinjury, which indicates that ADAMTS-7 facilitated intimal hyperplasia through degradation of inhibitory matrix protein COMP. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and postangioplasty restenosis. Key Words: metalloproteinase Ⅲ vascular smooth muscle cell migration Ⅲ neointima formation Ⅲ extracellular matrix M edia-to-intima migration of vascular smooth muscle cells (VSMCs) is pivotal to intimal thickening in atherosclerosis, restenosis after coronary angioplasty, and late failure of vein grafting. 1 Normally VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts as a barrier to VSMC migration. ECM degradation and remodeling require the activation of extracellular proteases, which in turn facilitate VSMC migration. 2 Previous studies have emphasized potential roles for the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP; the serine proteinases plasminogen activator and plasminogen; and the cysteine proteinases cathepsins K, L, and S during matrix remodeling and VSMC migration. 3 However, the identity of the matrix-degrading proteinases during pathological vascular remodeling in vivo has remained the subject of speculation.The recently identified metalloproteinase family of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) also degrade ECM. First identified in 1997, ADAMTS already showed strong biological relevance. 4 For example, ADAMTS...

Autophagy is a dynamic and highly regulated process of self-digestion responsible for cell survival and reaction to oxidative stress. As oxidative stress is increased in uremia and is associated with vascular calcification, we studied the role of autophagy in vascular calcification induced by phosphate. In an in vitro phosphate-induced calcification model of vascular smooth muscle cells (VSMCs) and in an in vivo model of chronic renal failure, autophagy was inhibited by the superoxide dismutase mimic MnTMPyP, superoxide dismutase-2 overexpression, and by knockdown of the sodium-dependent phosphate cotransporter Pit1. Although phosphate-induced VSMC apoptosis was reduced by an inhibitor of autophagy (3-methyladenine) and knockdown of autophagy protein 5, calcium deposition in VSMCs was increased during inhibition of autophagy, even with the apoptosis inhibitor Z-VAD-FMK. An inducer of autophagy, valproic acid, decreased calcification. Furthermore, 3-methyladenine significantly promoted phosphate-induced matrix vesicle release with increased alkaline phosphatase activity. Thus, autophagy may be an endogenous protective mechanism counteracting phosphate-induced vascular calcification by reducing matrix vesicle release. Therapeutic agents influencing the autophagic response may be of benefit to treat aging or disease-related vascular calcification and osteoporosis.

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Hyperphosphatemia is the major risk factor associated with vascular calcification (VC) in end-stage renal disease. As oxidative stress is increased in uremia, we studied the role of mitochondrial reactive oxygen species (ROS) and nuclear factor-κB signaling in phosphate-induced VC. In an in vitro calcification model (β-glycerophosphate (BGP) induction) using bovine aortic smooth muscle cells, the production of intracellular and mitochondrial ROS, or superoxide anion, was stimulated by increased mitochondrial membrane potential. This effect was blocked by the superoxide dismutase (SOD) mimic MnTMPyP, a respiratory chain inhibitor rotenone, or a protonophore. Calcium deposition and the switch of smooth muscle cells from a contractile to an osteogenic phenotype were decreased when mitochondrial ROS generation was inhibited by the respiratory chain inhibitor, MnTMPyP, or the overexpression of SOD1 and SOD2 and uncoupling protein 2. The phosphorylation of IkKβ, IκBα degradation, and p65 nuclear translocation were increased by BGP but reversed when mitochondrial ROS production was blocked by protonophore or MnTMPyP. Knockdown of endogenous p65 or overexpression of IκBα reduced calcium deposition in the cultured cells. Furthermore, in a rat model of dietary adenine-induced chronic renal failure, MnTMPyP reduced aortic ROS levels, p65 activation, and calcium deposition. Thus, mitochondrial ROS-mediated p65 nuclear translocation is involved in phosphate-induced VC.

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factorlike repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn 2؉ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.More than 15% of the world population older than 18 years are affected by arthritic disorders, including osteoarthritis (OA) 3 and rheumatoid arthritis (RA) (1). Accumulating evidence suggests that proteases perform an important function in the breakdown of the extracellular matrix in OA and RA (2). Cartilage oligomeric matrix protein (COMP), a prominent noncollagenous component of cartilage, accounts for ϳ1% of the wet weight of articular tissue (3, 4). COMP is a 524-kDa pentameric, disulfide-bonded, multidomain glycoprotein composed of approximately equal subunits (ϳ110 kDa each) (5, 6). Several studies suggest that monitoring of COMP levels (in both joint fluid and serum) can be used to assess the presence and progression of arthritis (7-11). Synovial fluid COMP levels were found to be higher in individuals with knee pain or injury (12), anterior cruciate ligament or meniscal injury (9, 12), OA (8, 12), and RA (7, 13) than in healthy individuals.Fragments of COMP have been detected in the cartilage, synovial fluid, and serum of patients with post-traumatic and primary OA and RA (7,8,13). The molecular mechanism of COMP degradation and the enzyme (s) responsible for it, however, remain largely unknown. Theoretically, inhibition of degradative enzymes can slow down or block the initiation and progression of arthritic diseases. The isolation of cartilage degradative enzymes is therefore of great interest from both a pathophysiological and a therapeutic standpoint. The ADAMTS family (ADAMTS: (a disintegrin and metalloprotease with thrombospondin motifs) consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least o...