Monocyte chemoattractant protein (MCP)–3 is inactivated upon cleavage by the matrix metalloproteinase (MMP) gelatinase A (MMP-2). We investigated the susceptibility to proteolytic processing of the 4 human MCPs by 8 recombinant MMPs to determine whether MCP-3 is an isolated example or represents a general susceptibility of chemokines to proteolytic inactivation by these important inflammatory proteases. In addition to MMP-2, MCP-3 is efficiently cleaved by membrane type 1 (MT1)–MMP, the cellular activator of MMP-2, and by collagenase-1 and collagenase-3 (MMP-1, MMP-13) and stromelysin-1 (MMP-3). Specificity was shown by absence of cleavage by matrilysin (MMP-7) and the leukocytic MMPs neutrophil collagenase (MMP-8) and gelatinase B (MMP-9). The closely related chemokines MCP-1, MCP-2, and MCP-4 were not cleaved by MMP-2 or MT1-MMP, but were cleaved by MMP-1 and MMP-3 with varying efficiency. MCPs were typically cleaved between residues 4 and 5, but MCP-4 was further processed at Val7-Pro8. Synthetic MCP analogs corresponding to the MMP-cleaved forms bound CC chemokine receptor (CCR)–2 and CCR-3, but lacked chemoattractant activity in pre-B cells transfected with CCR-2 and CCR-3 or in THP-1 monocytic cells, a transformed leukemic cell line. Moreover, the truncated products of MCP-2 and MCP-4, like MCP-3, were potent antagonists of their cognate CC chemokine receptors in transwell cell migration assays in vitro. When they were injected 24 hours after the initiation of carrageenan-induced inflammation in rat paws, their in vivo antagonist activities were revealed by a greater than 66% reduction in inflammatory edema progression after 12 hours. We propose that MMPs have an important role in modulating inflammatory and immune responses by processing chemokines in wound healing and in disease.

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. ) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein. X-linked retinoschisis (XLRS)1 is a common, inherited macular degeneration that affects males early in life (1-3). Affected individuals show a significant loss in central and in some cases peripheral vision, a splitting of the inner layers of the retina, and a loss in the b-wave of the electroretinogram. The gene responsible for XLRS was identified by positional cloning and shown to encode a retinal-specific 224-amino acid protein, known as RS1 or retinoschisin, containing a discoidin domain (4).RS1 is expressed and secreted from photoreceptor cells of the outer retina and bipolar cells of the inner retina as a multisubunit protein (5-8). The secreted protein associates with the surface of rod and cone photoreceptors at the level of the inner segment, outer nuclear, and outer plexiform layers and the surface of bipolar cells within the inner nuclear and inner plexiform layers of the retina. Biochemical studies further show that RS1 is tightly associated with the membrane fraction of retinal cell homogenates (6). RS1 is generally believed to function as a retinal cell adhesion protein, because mice deficient in RS1 have a highly disorganized retina with displacement of bipolar cells into the outer retinal layer, gaps between bipolar cells within the inner retina, disruption of the photoreceptor-bipolar synapse, and progressive degeneration of rod and cone photoreceptors (9).The dominant structural feature of the RS1 polypeptide is the 157-amino acid discoidin domain, also known as an F5/8 type C domain, which comprises over 75% of the processed polypeptide chain (4). Discoidin domains are present in a wide range of membrane and extracellular proteins where they mediate a variety of cell adhesion and cell signaling processes (10,11). Some proteins that contain discoidin domains are Factors V and VIII involved in blood coagulation, neuropilins 1 and 2, which mediate nervous system regeneration and degeneration, discoidin domain receptors implicated in cancer meta...

Angiotensin II (Ang II) resets the baroreflex control of heart rate to a higher blood pressure. This action is apparently mediated via Ang II receptors in the area postrema, but it is not known if these are of the AT, or AT2 subtype. In the present study the effects of losartan, a selective AT, receptor antagonist, and PD 123319, a selective AT2 antagonist, on the cardiac baroreflex response to Ang II were investigated in conscious rabbits with chronically implanted arterial and venous catheters. Baroreflex curves were generated with intravenous infusions of phenylephrine and nitroprusside (2.6-25 $ug/kg per min) and analyzed using a four-parameter logistic model to yield their upper and lower plateaus, arterial pressure at the midpoint of the heart rate range (BP50), and slope coefficient.From these four parameters, the gain and range of the baroreflex were calculated. Background intravenous infusion of Ang II at 10 ng/kg per min increased mean arterial pressure by 17 mmHg but did not change heart rate. Ang II shifted the baroreflex curve to the right as indicated by an increase in BP5o from 70.9±2.0 to 89.3±2.7 mmHg (P < 0.05), but did not change baroreflex gain significantly. Ang II did not alter the upper plateau of the baroreflex, but decreased the lower plateau from 119.4±10.3 to 73.6±11.5 beats per minute (bpm) (P < 0.05), extending the heart rate range by 52.5 bpm. Pretreatment with losartan completely abolished the pressor and cardiac baroreflex responses to Ang II. In contrast, PD 123319 had no effect on these responses. Administration of losartan alone to block endogenous Ang II shifted the baroreflex curve to the left as indicated by a decrease in BP5o from 71.2±2.7 to 64.7±2.5 mmHg (P < 0.05). These results demonstrate that the resetting of the baroreflex control of heart rate by Ang II is mediated by AT1 receptors, and that basal levels of endogenous Ang II exert a tonic action on the cardiac baroreflex to increase the setpoint around which the baroreflex regulates heart rate. (J.

G-protein-coupled receptors (GPCRs) and other, structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here the implementation of an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tagspecific, high-affinity monoclonal antibody. Expressing two GPCRs as well as two related ATP binding cassette (ABC) transporters in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 in an annotated spectrum library, and to identify the N-glycosylation sites for CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.

Purpose This study aims to identify clinical factors that may predispose struvite stone patients to urosepsis following percutaneous nephrolithotomy (PCNL). Materials and Methods A retrospective review was conducted on patients who received PCNL for struvite stones. The Systemic Inflammatory Response Syndrome (SIRS) criteria and quick-Sepsis Related Organ Failure Assessment (q-SOFA) criteria were used to identify patients who were at an increased risk for urosepsis. Statistical analysis was performed using Fisher's exactness test, Wilcoxon rank test, and logistic regression. Results Chart review identified 99 struvite stone patients treated with PCNL. Post-operatively, 40 patients were SIRS positive (≥2 criteria) and/or q-SOFA positive (score ≥2). Using SIRS as an approximation for urosepsis, longer operative times (p<0.001), higher pre-operative white blood cell counts (p=0.01), greater total stone surface area (p<0.0001), and pre-operative stenting (OR, 5.75; p=0.01) were identified as independent risk factors for urosepsis. Multivariate analysis demonstrated pre-operative stenting (OR, 1.46; p=0.01) to be a risk factor. With q-SOFA, univariable analysis found that antibiotic use within 3 months prior to a PCNL (OR, 4.44; p=0.04), medical comorbidities (OR, 4.80; p=0.02), longer operative times (p<0.001), lengthier post-operative hospitalization (p<0.01), and greater total stone surface area (p<0.0001) were risk factors for urosepsis. Multivariate analysis revealed that bladder outlet obstruction (OR, 2.74; p<0.003) and pre-operative stenting (OR, 1.27; p=0.01) significantly increased odds of being q-SOFA positive. Conclusions Several risk factors for urosepsis following PCNL for struvite stones have been identified. These risk factors should be taken into consideration in peri-operative care to mitigate the risks of urosepsis.