Rationale: SM22 (or transgelin), an actin-binding protein abundant in vascular smooth muscle cells (VSMCs), is downregulated in atherosclerosis, aneurysm and various cancers. Abolishing SM22 in apolipoprotein E knockout mice accelerates atherogenesis. However, it is unclear whether SM22 disruption independently promotes arterial inflammation. Objective: To investigate whether SM22 disruption directly promotes inflammation on arterial injury and to characterize the underlying mechanisms. Methods and Results: Using carotid denudation as an artery injury model, we showed that Sm22 knockout (Sm22 ؊/؊ ) mice developed enhanced inflammatory responses with higher induction of proinflammatory genes, including Vcam1, Icam1, Cx3cl1, Ccl2, and Ptgs2. Higher expression of these genes was confirmed in primary Sm22 ؊/؊ VSMCs and in PAC1 cells after Sm22 knockdown, whereas SM22 recapitulation in primary Sm22 ؊/؊ VSMCs decreased their expression. NFKB2 was prominently activated in both injured carotids of Sm22 ؊/؊ mice and in PAC1 cells after Sm22 knockdown and may mediate upregulation of these proinflammatory genes. As a NF-B activator, reactive oxygen species (ROS) increased in primary Sm22 ؊/؊ VSMCs and in PAC1 cells after Sm22 knockdown. ROS scavengers blocked NF-B activation and induction of proinflammatory genes. Furthermore, Sm22 knockdown increased Sod2 expression and activated p47phox, reflecting contributions of mitochondria and NADPH oxidase to the augmented ROS production; this may result from actin and microtubule cytoskeletal remodeling. Conclusions: Our findings show that SM22 downregulation can induce proinflammatory VSMCs through activation of ROS-mediated NF-B pathways. This study provides initial evidence linking VSMC cytoskeleton remodeling with arterial inflammation. (Circ Res. 2010;106:1351-1362.)Key Words: VSMC Ⅲ SM22 Ⅲ inflammation Ⅲ NF-B Ⅲ ROS S M22, also known as SM22␣ or transgelin, is a 22 kDa protein abundant in the smooth muscle cells (SMCs) of vertebrates. 1 It belongs to the calponin family because it contains an N-terminal calponin homology domain and a C-terminal calponin-like domain. 2 The basic molecular function of SM22 is to bind actin and facilitate the formation of cytoskeletal structures such as stress fibers. 2 SM22 dysregulation is observed in a variety of human diseases. For instance, expression of SM22 is decreased in several types of cancer. 3 Expression of SM22 is also downregulated in atherosclerotic arteries 4,5 and abdominal aortic aneurysms. 6 These findings suggest a correlation between decreased SM22 expression and arterial diseases. However, it is unclear whether the SM22 downregulation promotes the pathogenesis of arterial diseases or whether it is simply a passive outcome.SM22 has been widely used as a SMC marker during embryogenesis and in adult. 7 Sm22 knockout (Sm22 Ϫ/Ϫ ) mice are viable and fertile with uncompromised vasculature development. 8 -10 This suggests that SM22 may be either functionally redundant or compensated during vasculature development. However, th...

Substrates of the ubiquitin system are degraded by the 26 S proteasome, a complex protease consisting of at least 32 different subunits. Recent studies showed that RPN4 (also named SON1 and UFD5) is a transcriptional activator required for normal expression of the Saccharomyces cerevisiae proteasome genes. Interestingly, RPN4 is extremely short-lived and degraded by the 26 S proteasome, establishing a feedback circuit that controls the homeostatic abundance of the 26 S proteasome. The mechanism underlying the degradation of RPN4, however, remains unclear. Here we demonstrate that the proteasomal degradation of RPN4 is mediated by two independent degradation signals (degron). One degron leads to ubiquitylation on internal lysine(s), whereas the other is independent of ubiquitylation. Stabilization of RPN4 requires inhibition of internal ubiquitylation and inactivation of the ubiquitin-independent degron. RPN4 represents the first proteasomal substrate in S. cerevisiae that can be degraded through ubiquitylation or without prior ubiquitylation. This finding makes it possible to use both yeast genetics and biochemical analysis to investigate the mechanism of ubiquitin-independent proteolysis.The ubiquitin-proteasome system is the primary intracellular machinery responsible for elimination of abnormal proteins and selective destruction of regulatory proteins involved in a wide range of cellular processes (1-4). Substrates of the 26 S proteasome are normally degraded via ubiquitylation on internal lysines. Several lines of evidence from our early work, however, suggested that ubiquitylation might not be required for the degradation of RPN4, a transcriptional activator of the S. cerevisiae proteasome genes (5, 6). First, the degradation of RPN4 was not noticeably impaired in the uba1-2 mutant, which underexpresses the E1 1 enzyme, and is therefore deficient in ubiquitylation of proteins (7). Second, overexpression of Ub K48R,G76A , a ubiquitin mutant that inhibits the formation of lysine 48-linked multiubiquitin chains (8), did not significantly decrease the turnover rate of RPN4. Third, the half-life of RPN4 in wild type cells was comparable with that in the mutants that lack one of the known E2 enzymes or one of the following E3 enzymes: UBR1, UFD4, RSP5, TOM1, HUL4 or HUL5 (9 -12). In the present study, we explore the mechanism underlying the degradation of RPN4. Strikingly, we found that the proteasomal degradation of RPN4 can be mediated by two independent degrons. One involves ubiquitylation on internal lysine(s), whereas the other is apparently ubiquitin-independent. Stabilization of RPN4 requires inhibition of both degrons. This finding demonstrates that the same proteasomal substrate can be degraded dependently or independently of ubiquitylation. Ref. 14). Details of plasmid constructs are available upon request. Lysine-to-arginine substitutions were constructed by PCR-mediated site-directed mutagenesis and confirmed by DNA sequencing. For pulse-chase analysis, RPN4 and truncated derivatives were expressed ...

The homeostatic abundance of the proteasome in Saccharomyces cerevisiae is controlled by a feedback circuit in which transcriptional activator Rpn4 up-regulates the proteasome genes and is destroyed by the assembled, active proteasome. Remarkably, the degradation of Rpn4 can be mediated by two independent pathways. One pathway is independent of ubiquitin, whereas the other involves ubiquitination on internal lysines. In the present study, we investigated the mechanism underlying the ubiquitin-dependent degradation of Rpn4. We demonstrated, through in vivo and in vitro assays, that Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase, which was originally identified as a sequence homolog of Ubr1, the E3 component of the N-end rule pathway. The ubiquitin-conjugating enzyme Rad6, which directly interacts with Ubr2, is also required for the ubiquitin-dependent degradation of Rpn4. Furthermore, we showed that deletion of UBR2 exhibited a strong synthetic growth defect with a mutation in the Rpt1 proteasome subunit when Rpn4 was overexpressed. This study not only identified the ubiquitination apparatus for Rpn4 but also unveiled the first physiological substrate of Ubr2. The biological significance of Ubr2-mediated degradation of Rpn4 is also discussed.The ubiquitin (Ub) 1 proteasome system is the primary intracellular machinery responsible for elimination of abnormal proteins and selective destruction of regulatory proteins involved in a wide variety of cellular processes (1-3). Ubiquitination of a protein substrate is a consecutive process involving multiple enzymes (4). Ub is first activated by the Ub-activating enzyme (E1), forming a thioester between the C-terminal carboxyl group of Ub and a specific cysteine of the E1. The Ub moiety of the E1ϳUb thioester is thereafter transferred to one of the Ub-conjugating enzymes (E2). The Ub moiety of the E2ϳUb thioester is conjugated via an isopeptide bond to the ⑀-amino group of a lysine residue of a substrate or a preceding Ub molecule conjugated to the substrate, the latter reaction resulting in a substrate-linked multi-Ub chain. Most E2s function in complex with one of the E3 enzymes or Ub ligases. A Ub ligase also denotes an E2⅐E3 complex. Ubiquitination of a specific substrate is regulated through modulation of its degradation signal and through control of the activity of a cognate E3 (4 -9). The isopeptide bond between Ub and a substrate can be hydrolyzed by deubiquitinating enzymes, which provides yet another layer of regulation for substrate ubiquitination (10).Most known E3s are grouped into two families (homology to E6-AP C terminus domain E3s and RING E3s) based on their catalytic modules and features of sequence and structure (4, 9). A homology to E6-AP C terminus-domain E3 can accept Ub moiety from an associated E2ϳUb thioester, forming an E3ϳUb thioester and acting as a proximal Ub donor to the substrate that it selects. By contrast, formation of thioesters between RING E3s and Ub has not been detected. The precise mechanism of RING E3-mediated ubi...