Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Regulation of CED-3 caspase localization and activation by C. elegans nuclear-membrane protein NPP-14.

Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that the CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear-pore protein NPP-14 interacts with the CED-3 zymogen prodomain, colocalizes with CED-3 in vivo and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and decreased CED-3 activation by CED-4 in the presence of nuclei or NPP-14, thus leading to cell-death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role of nuclei and nuclear-membrane proteins in regulating the activation and localization of CED-3.

Evidence for Extending the Duration of Chemoprophylaxis following Free Flap Harvest from the Lower Extremity: Prospective Screening for Deep Venous Thrombosis.

BACKGROUND: The purpose of this study was to investigate the incidence of symptomatic and asymptomatic deep venous thrombosis in patients undergoing harvest of a free flap from the lower extremity who were receiving standard chemoprophylaxis while hospitalized. METHODS: A retrospective review of 65 consecutive patients undergoing surgery between 2011 and 2013 was performed to determine the incidence of symptomatic deep venous thrombosis. These patients were screened for deep venous thrombosis based on development of symptoms. Prospective evaluation of a similar consecutive population of 37 patients between 2014 and 2015 was then performed to determine the incidence of asymptomatic deep venous thrombosis. These patients underwent routine duplex ultrasonography of both legs at postoperative weeks 1 and 4. RESULTS: Symptomatic deep venous thrombosis occurred in 2.9 percent of all patients. In the prospective cohort, 8.1 percent of the patients were found to have an acute deep venous thrombosis by postoperative week 1. At postoperative week 4, 16.7 percent of the patients developed a new, acute deep venous thrombosis. The estimated costs of screening and treating deep venous thrombosis in the retrospective group and the prospective group were $222 and $2259, respectively. The cost of routine chemoprophylaxis without duplex screening for an additional 14 days after discharge was $125 per patient. CONCLUSIONS: The rate of asymptomatic deep venous thrombosis may be much higher than previously appreciated in this population of very high-risk patients, especially during the 2 weeks after discharge. Extending the duration of chemoprophylaxis to 4 weeks after surgery may be warranted. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

The impact of congenital cardiovascular malformations on the assessment and surgical management of infants with cleft lip and/or palate.

OBJECTIVE: The purpose of this study was to assess the cardiac evaluation of cleft lip and/or palate patients, characterize their cardiovascular malformations, and determine the impact of cardiovascular malformations on surgical management. DESIGN: A single-institution retrospective study of 329 consecutive cleft patients was performed. Cardiovascular malformations were categorized according to involvement of cardiac septa, vasculature, and valves. Their impact on the need for cardiac surgery, timing of cleft repair, need for subacute bacterial endocarditis (SBE) prophylaxis, and the perioperative experience was evaluated. RESULTS: Ten percent (33/329) of cleft patients had a cardiovascular malformation, and 3% underwent cardiac surgery prior to cleft repair. Malformations of the septa, vasculature, and valves were present in 9%, 6%, and 2% of cleft infants, respectively. Murmur as a sign of structural cardiovascular disease was 79% sensitive and 97% specific. Cleft palate repair was delayed by 2 months in patients with a cardiovascular malformation (P = .001). Subacute bacterial endocarditis prophylaxis was recommended, not recommended, or not specified by cardiology in 18%, 33%, and 48% of cleft patients with a cardiovascular malformation, respectively. Postoperative stay and surgical complications were not associated with cardiovascular malformation. CONCLUSIONS: Even in the absence of a murmur, echocardiographic screening should be considered in infants with nonspecific signs of cardiovascular disease. Greater awareness of the guidelines for SBE prophylaxis is needed. Most cleft patients with a cardiovascular malformation do not require cardiac surgery and do not experience an increased rate of complications associated with cleft surgery.

Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction.

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans.

HBx is a multifunctional hepatitis B virus (HBV) protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. However, the host targets and mechanisms of action of HBx are poorly characterized. We show here that expression of HBx in Caenorhabditis elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV. Genetic and biochemical analyses indicate that HBx interacts directly with the B-cell lymphoma 2 (Bcl-2) homolog CED-9 (cell death abnormal) through a Bcl-2 homology 3 (BH3)-like motif to trigger both cytosolic Ca(2+) increase and cell death. Importantly, Bcl-2 can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, suggesting that CED-9 and Bcl-2 are conserved cellular targets of HBx. A genetic suppressor screen of HBx-induced cell death has produced many mutations, including mutations in key regulators from both apoptosis and necrosis pathways, indicating that this screen can identify new apoptosis and necrosis genes. Our results suggest that C. elegans could serve as an animal model for identifying crucial host factors and signaling pathways of HBx and aid in development of strategies to treat HBV-induced liver disorders.

JAK2 inhibition for the treatment of hematologic and solid malignancies.

INTRODUCTION: Mutations in Janus kinase 2 (JAK2), and in particular JAK2 V617F, are common in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In the past several years, JAK2 inhibitors have been rapidly developed as targeted therapies for MPNs. AREAS COVERED: JAK2 mutations, including JAK2 V617F and unique fusion proteins, are critical for oncogenesis of some hematologic malignancies. Although JAK2 mutations are extremely rare in solid cancers, pathophysiological JAK2/STAT signaling can still promote tumor cell growth, proliferation, migration, invasion and angiogenesis. JAK2 inhibition can curtail malignant cellular behaviors and thus may be a promising therapeutic strategy. EXPERT OPINION: The involvement of oncogenic JAK2 mutations in hematologic malignancies indicates that JAK2 inhibition has the potential to be a highly successful treatment option. The exact role of JAK2 signaling in solid cancers is unclear, but JAK2 inhibition may prevent disease progression by restricting malignant cell phenotypes. JAK2 inhibitors in development for the treatment of MPNs have demonstrated clinical activity with minimal toxicity. This class of agents should be investigated more rigorously for the treatment of other malignancies with aberrant JAK2 signaling with or without JAK2 mutations.

Improved survival and reduced vascular permeability by eliminating or blocking 12/15-lipoxygenase in mouse models of acute lung injury (ALI).

Acute lung injury (ALI) is a prevalent disease associated with high mortality. 12/15-lipoxygenase (12/15-LO) is an enzyme producing 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE from arachidonic acid. To test whether 12/15-LO is involved in increasing vascular permeability in the lung, we investigated the role of 12/15-LO in murine models of LPS-induced pulmonary inflammation and clinically relevant acid-induced ALI. The vascular permeability increase upon LPS inhalation was abolished in Alox15(-/-) mice lacking 12/15-LO and in wild-type mice after pharmacological blockade of 12/15-LO. Alox15(-/-) mice also showed improved gas exchange, reduced permeability increase, and prolonged survival in the acid-induced ALI model. Bone marrow chimeras and reconstitution experiments revealed that 12-HETE produced by hematopoietic cells regulates vascular permeability through a CXCR2-dependent mechanism. Our findings suggest that 12/15-LO-derived 12-HETE is a key mediator of vascular permeability in acute lung injury.

Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4.

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.

Endothelial cell PECAM-1 promotes atherosclerotic lesions in areas of disturbed flow in ApoE-deficient mice.

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis. METHODS AND RESULTS: We crossed C57BL/6 Pecam1(-/-) mice with apolipoprotein E-deficient (Apoe(-/-)) mice. On a Western diet, Pecam1(-/-)Apoe(-/-) mice showed reduced atherosclerotic lesion size compared to Apoe(-/-) mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-kappaB were all reduced in Pecam1(-/-)Apoe(-/-) mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-kappaB activity and VCAM-1 expression under conditions of atheroprone flow. CONCLUSIONS: These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-kappaB-mediated gene expression.

CXCR6 promotes atherosclerosis by supporting T-cell homing, interferon-gamma production, and macrophage accumulation in the aortic wall.

BACKGROUND: T lymphocytes are thought to be important in atherosclerosis, but very little is known about the mechanisms of lymphocyte recruitment into atherosclerosis-prone aortas. In this study we tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of CD4+ T helper 1 cells and natural killer T cells, is involved in lymphocyte homing into the aortic wall and modulates the development and progression of atherosclerosis. METHODS AND RESULTS: To investigate the role of CXCR6 in the development and progression of atherosclerosis, we bred CXCR6-deficient (CXCR6(GFP/GFP)) mice with apolipoprotein E-deficient (ApoE(-/-)) mice. We found that CXCR6(GFP/GFP)/ApoE(-/-) mice fed a Western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE(-/-) controls. Flow cytometry analysis of the aortas from CXCR6(GFP/GFP)/ApoE(-/-) mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas resulted in significantly diminished production of interferon-gamma and reduction of CD11b+/CD68+ macrophages in the aorta. CONCLUSIONS: These data provide evidence for a proatherosclerotic role of CXCR6. Absence of CXCR6 alters the recruitment of CXCR6+ leukocytes and modulates the local immune response within the aortic wall.

Critical role of macrophage 12/15-lipoxygenase for atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Mice lacking leukocyte type 12/15-lipoxygenase (12/15-LO) show reduced atherosclerosis in several models. 12/15-LO is expressed in a variety of cells, including vascular cells, adipocytes, macrophages, and cardiomyocytes. The purpose of this study was to determine which cellular source of 12/15-LO is important for atherosclerosis. METHODS AND RESULTS: Bone marrow from 12/15-LO-/-/apoE-/- mice was transplanted into apoE-/- mice and vice versa. Deficiency of 12/15-LO in bone marrow cells protected apoE-/- mice fed a Western diet from atherosclerosis to the same extent as complete absence of 12/15-LO, although plasma 8,12-iso-iPF2alpha-IV, a measure of lipid peroxidation, remained elevated. 12/15-LO-/-/apoE-/- mice regained the severity of atherosclerotic lesion typical of apoE-/- mice after replacement of their bone marrow cells with bone marrow from apoE-/- mice. Peritoneal macrophages obtained from wild-type but not 12/15-LO-/- mice caused endothelial activation in the presence of native LDL. Absence of 12/15-LO decreased the ability of macrophages to form foam cells when exposed to LDL. CONCLUSIONS: We conclude that macrophage 12/15-LO plays a dominant role in the development of atherosclerosis by promoting endothelial inflammation and foam cell formation.