Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.

Elastic fibers are components of the extracellular matrix and confer resilience. Once laid down, they are thought to remain stable, except in the uterine tract where cycles of active remodeling occur. Loss of elastic fibers underlies connective tissue aging and important diseases including emphysema. Failure to maintain elastic fibers is explained by a theory of antielastase-elastase imbalance, but little is known about the role of renewal. Here we show that mice lacking the protein lysyl oxidase-like 1 (LOXL1) do not deposit normal elastic fibers in the uterine tract post partum and develop pelvic organ prolapse, enlarged airspaces of the lung, loose skin and vascular abnormalities with concomitant tropoelastin accumulation. Distinct from the prototypic lysyl oxidase (LOX), LOXL1 localizes specifically to sites of elastogenesis and interacts with fibulin-5. Thus elastin polymer deposition is a crucial aspect of elastic fiber maintenance and is dependent on LOXL1, which serves both as a cross-linking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.

Myosin accumulation and striated muscle myopathy result from the loss of muscle RING finger 1 and 3.

Maintenance of skeletal and cardiac muscle structure and function requires precise control of the synthesis, assembly, and turnover of contractile proteins of the sarcomere. Abnormalities in accumulation of sarcomere proteins are responsible for a variety of myopathies. However, the mechanisms that mediate turnover of these long-lived proteins remain poorly defined. We show that muscle RING finger 1 (MuRF1) and MuRF3 act as E3 ubiquitin ligases that cooperate with the E2 ubiquitin-conjugating enzymes UbcH5a, -b, and -c to mediate the degradation of beta/slow myosin heavy chain (beta/slow MHC) and MHCIIa via the ubiquitin proteasome system (UPS) in vivo and in vitro. Accordingly, mice deficient for MuRF1 and MuRF3 develop a skeletal muscle myopathy and hypertrophic cardiomyopathy characterized by subsarcolemmal MHC accumulation, myofiber fragmentation, and diminished muscle performance. These findings identify MuRF1 and MuRF3 as key E3 ubiquitin ligases for the UPS-dependent turnover of sarcomeric proteins and reveal a potential basis for myosin storage myopathies.

Selective identification of secreted and transmembrane breast cancer markers using Escherichia coli ampicillin secretion trap.

Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST). For CAST, we constructed a plasmid in which the signal sequence of beta-lactamase was deleted such that it does not confer ampicillin resistance. Eukaryotic cDNA libraries cloned into pCAST produced tens of thousands of ampicillin-resistant clones, 80% of which contained cDNA fragments encoding secreted and membrane spanning proteins. We identified 2,708 unique sequences from cDNA libraries made from surgical breast cancer specimens. We analyzed the expression of 1,287 of the 2,708 genes and found that 166 were overexpressed in breast cancers relative to normal breast tissues. Eighty-five percent of these genes had not been previously identified as markers of breast cancer. Twenty-three of the 166 genes (14%) were relatively tissue restricted, suggesting use as cancer-specific targets. We also identified several new markers of ovarian cancer. Our results indicate that CAST is a robust, rapid, and low cost method to identify cell surface and secreted proteins and is applicable to a variety of relevant biological questions.

Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction.

RING-finger proteins commonly function as ubiquitin ligases that mediate protein degradation by the ubiquitin-proteasome pathway. Muscle-specific RING-finger (MuRF) proteins are striated muscle-restricted components of the sarcomere that are thought to possess ubiquitin ligase activity. We show that mice lacking MuRF3 display normal cardiac function but are prone to cardiac rupture after acute myocardial infarction. Cardiac rupture is preceded by left ventricular dilation and a severe decrease in cardiac contractility accompanied by myocyte degeneration. Yeast two-hybrid assays revealed four-and-a-half LIM domain (FHL2) and gamma-filamin proteins as MuRF3 interaction partners, and biochemical analyses showed these proteins to be targets for degradation by MuRF3. Accordingly, FHL2 and gamma-filamin accumulated to abnormal levels in the hearts of mice lacking MuRF3. These findings reveal an important role of MuRF3 in maintaining cardiac integrity and function after acute myocardial infarction and suggest that turnover of FHL2 and gamma-filamin contributes to this cardioprotective function of MuRF3.

Abnormal striatal dopaminergic synapses in National NeuroAIDS Tissue Consortium subjects with HIV encephalitis.

People with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) have neurological problems that overlap with diseases associated with abnormal dopaminergic (DAergic) synaptic transmission, including subcortical dementia, motor slowing, psychosis, and drug addiction. Previous study has suggested that DAergic tone may be decreased in HIV/AIDS, but biochemical confirmation of that tenet is still lacking. To that end, this study addresses the neurochemical interaction between HIV infection and DAergic synaptic transmission in human brain specimens. Protein markers of DAergic synapses were characterized in homogenates of the corpus striatum from individuals with HIV encephalitis (HIVE) and seronegative controls from the autopsy cohort of the National NeuroAIDS Tissue Consortium. Striatal DAergic markers were abnormal in HIVE. Abnormal presynaptic markers included decreased tyrosine hydroxylase (TH) protein and decreased phosphorylated TH. The presynaptic dopamine reuptake transporter (DAT) was increased reciprocally. Postsynaptic abnormalities included decreased dopamine receptor type 2 (D(2)R) and increased D(3)R. There was preferential loss of the alternatively spliced long isoform of D(2)R relative to the short isoform. Abnormal DAergic synapse proteins were significantly correlated with the HIV Gag mRNA transcripts amplified in striatal extracts. These synaptic changes resemble shifts that occur when DAergic tone is increased experimentally. Increased DAergic tone leads to heightened salience for drugs of abuse, increases behaviors that increase the risk of HIV transmission, and might decrease compliance with antiretroviral medication regimens.

Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration.

Fibulin (fbln)-5 is an elastin-binding protein required for assembly and organization of elastic fibers. To examine the potential role of fbln-5 in vascular remodeling and neointima formation, we induced vascular injury by carotid artery ligation in fbln-5(-/-) mice. Mutant mice displayed an exaggerated vascular remodeling response that was accompanied by severe neointima formation with thickened adventitia. These abnormalities were not observed in elastin(+/-) mice that exhibited a comparable reduction of vessel extensibility to fbln-5(-/-) mice. Thus, the severe remodeling response could not be attributed to altered extensibility of the vessel wall alone. Vascular smooth muscle cells cultured from fbln-5(-/-) mice displayed enhanced proliferative and migratory responses to mitogenic stimulation relative to wild-type cells, and these responses were inhibited by overexpression of fbln-5. These findings demonstrate the importance of the elastic laminae in vascular injury, and reveal an unexpected role of fbln-5 as an inhibitor of vascular smooth muscle cell proliferation and migration.

STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription.

Changes in actin dynamics influence diverse cellular processes and couple the actin-based cytoskeleton to changes in gene transcription. Members of the Rho GTPase family regulate cytoskeletal organization by stimulating actin polymerization and stress fiber formation when activated by extracellular signaling. The transcriptional activity of serum response factor (SRF) is stimulated in response to changes in actin dynamics and Rho signaling, but the proteins that mediate this phenomenon have not been fully identified. We describe a novel, evolutionarily conserved actin-binding protein, called STARS (striated muscle activator of Rho signaling), that is expressed specifically in cardiac and skeletal muscle cells. STARS binds to the I-band of the sarcomere and to actin filaments in transfected cells, where it activates Rho-signaling events. STARS stimulates the transcriptional activity of SRF through a mechanism that requires actin binding and involves Rho GTPase activation. STARS provides a potential mechanism for specifically enhancing Rho-dependent transcription in muscle cells and for linking changes in actin dynamics to gene transcription.