Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.

Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1's ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.

The ERAD system is restricted by elevated ceramides.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

Five-year outcomes from a prospective, multicenter study of endovascular repair of iliac artery aneurysms using an iliac branch device.

OBJECTIVE: We have reported the 5-year results of a pivotal prospective, multicenter study conducted in the United States of a specifically designed iliac branch endoprosthesis (IBE; W.L. Gore & Associates, Flagstaff, AZ) for endovascular repair of aortoiliac aneurysms and common iliac artery aneurysms. METHODS: A total of 63 patients (98.4% male; mean age, 70 years) with aortoiliac or common iliac artery aneurysms had undergone implantation of a single IBE device and a bifurcated aortoiliac stent graft. Patients with bilateral common iliac artery aneurysms (n = 22; 34.9%) had undergone either staged occlusion or surgical revascularization of the contralateral internal iliac artery before study enrollment. At 5 years, 36 of the 63 patients had completed the final study follow-up examinations, including clinical examinations (n = 35) and computed tomography (n = 32), with the results evaluated by an independent core laboratory and adverse events adjudicated by a clinical events committee. RESULTS: At 5 years, freedom from all-cause mortality was 85.7% and freedom from aneurysm-related mortality was 100%. The nine deaths that had occurred (range, 132-1898 days) were adjudicated as unrelated to the aneurysm or procedure. Primary patency of the internal and external iliac artery IBE limbs was 95.1% and 100%, respectively. No patients had experienced new-onset buttock claudication on the IBE side or self-reported new-onset erectile dysfunction. The common iliac artery diameter on the IBE side was either unchanged or had decreased by ≥5 mm in 30 of the 31 patients (96.8%) with a baseline (1 month) and 5-year (range, 1641-2006 days) computed tomography scan available. Of the 31 evaluable patients, 9 (29.0%) had had an increase of ≥5 mm in the aortic diameter, 5 of whom had had a concurrent type II endoleak. No type I or type III endoleaks or device migration were identified by the core laboratory. Six patients had undergone eight secondary interventions, including five interventions for a type II endoleak. The freedom from secondary intervention was 90.5%. CONCLUSIONS: The 5-year results of our prospective, multicenter study have confirmed the safety, efficacy, and durability of the IBE device for the treatment of aortoiliac and iliac artery aneurysms. The device effectively prevented common iliac artery aneurysm rupture, maintained the patency of the internal iliac artery, and avoided the complications associated with internal iliac artery sacrifice. Although common iliac artery aneurysm enlargement was rare, abdominal aortic enlargement was more common, suggesting that the outcomes of endovascular aneurysm repair might be different for patients with or without associated common iliac artery aneurysms.

Predictors of Aneurysm Sac Shrinkage Utilizing a Global Registry.

BACKGROUND: Aneurysm sac remodeling is a complex multifactorial process with unknown factors influencing sac regression after endovascular aortic aneurysm repair (EVAR). We sought to identify factors associated with this process by analyzing data obtained from patients treated with the GORE EXCLUDER endovascular aneurysm repair (EVAR) endoprosthesis from December 2010 to October 2016 enrolled in the Global Registry for Endovascular Aortic Treatment (GREAT). METHODS: All patients enrolled in GREAT with three years CT angiography (CTA) follow-up in each of the three successive years after EVAR were included. The percentage of sac size reduction toward device diameter was calculated and used as a surrogate for sac regression with the formula used being: sac size reduction = ((AAA baseline diameter - AAA diameter at follow-up)/(AAA baseline diameter - device diameter))∗100. The cohort was divided into two groups in accordance with the percentage of aneurysm sac reduction at three years; one with the top quartile of patients and the other with the lowest three quartiles. Demographic and procedural variables were analyzed using univariate and regression modeling to determine factors predictive of sac regression. RESULTS: There were 3265 subjects enrolled with follow-up as of May 2018 of which 526 (16.2%) had three years of CTA surveillance. Overall aneurysm sac size decreased from a mean of 58.0 mm (Std Dev: 10.4, range: 34.2, 100.0) to a mean of 49.3 mm (Std Dev: 14.1, range: 0, 140) for a percentage reduction toward device diameter of a mean 28.2% (Std Dev: 39.0, range: -103.7, 183.9). On multivariate logistic regression model; two factors proved to be statistically significant contributors to a larger percentage reduction in aneurysm sac: a conical neck (odds ratio [OR] = 1.64, P-value = 0.023) and a larger proximal device diameter (OR = 1.09, P-value = 0.023). On the other hand, two factors were negative predictors of sac shrinkage, namely: old age (OR = 0.96, P-value = 0.002) and larger baseline aneurysm sac diameter (OR = 0.98, P-value = 0.028). CONCLUSIONS: Aneurysms with conical necks and larger proximal device neck diameters have an increased percentage change in sac size over time after EVAR. Older age and larger initial diameters of aneurysms were negatively associated with percentage change in sac size as well as sac regression. Further study is needed to determine the clinical utility of these observations and applicability across multiple endoprosthesis platforms.

Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.

Human cells have defense mechanisms against viral infection known as restriction factors. These are proteins that break down parts of a virus including its DNA or proteins. To evade these defenses, viruses in turn make proteins that block or break down restriction factors. This battle between human and viral proteins determines which types of cells are infected and how quickly a virus can multiply and spread to new cells. HIV produces a protein called Vpr that counteracts a restriction factor found in immune cells called macrophages. However, the identity of the restriction factor targeted by Vpr is a mystery. When Vpr is missing, this unknown restriction factor breaks down a virus protein called Env. Env is a glycoprotein, which is a protein with sugars attached. When Env levels are low, HIV cannot spread to other cells and multiply. Identifying the restriction factor that breaks down Env may lead to new ways of treating and preventing HIV infections. Now, Lubow et al. reveal that the unknown restriction factor in macrophages is a protein called the mannose receptor. This protein binds and destroys proteins containing mannose, a type of sugar found on bacteria and some viruses. The experiments revealed that the mannose receptor grabs mannose on the HIV protein Env. This causes Env to be broken down and stops HIV from spreading. Lubow et al. also find that Vpr works with another protein produced by HIV called Nef to reduce the number of mannose receptors on macrophages. The two proteins do this by targeting different steps in the assembly of mannose receptors, allowing the virus to multiply and spread more efficiently. The experiments suggest that drugs that simultaneously block Vpr and Nef might prevent or suppress HIV infections. More studies are needed to develop and test potential HIV-treatments targeting Vpr and Nef.

Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1.

Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and polyubiquitinated before being degraded by the proteasome. The multi-spanning ubiquitin ligase Hrd1 forms the retrotranslocation channel and associates with three other membrane proteins (Hrd3, Usa1, Der1) of poorly defined function. The Hrd1 channel is gated by autoubiquitination, but how Hrd1 escapes degradation by the proteasome and returns to its inactive ground state is unknown. Here, we show that autoubiquitination of Hrd1 is counteracted by Ubp1, a deubiquitinating enzyme that requires its N-terminal transmembrane segment for activity towards Hrd1. The Hrd1 partner Hrd3 serves as a brake for autoubiquitination, while Usa1 attenuates Ubp1's deubiquitination activity through an inhibitory effect of its UBL domain. These results lead to a model in which the Hrd1 channel is regulated by cycles of autoubiquitination and deubiquitination, reactions that are modulated by the other components of the Hrd1 complex.

Doxorubicin as a fluorescent reporter identifies novel MRP1 (ABCC1) inhibitors missed by calcein-based high content screening of anticancer agents.

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum multidrug resistance and can decrease the bioavailability of many important drugs. Substrates of MRP1 include anti-cancer agents, antibiotics, antivirals, antidepressants and anti-inflammatory drugs. Using calcein as a fluorescent reporter in a high content uptake assay, we recently reported the identification of 12 MRP1 inhibitors after screening an anti-cancer library of 386 compounds. Here, we describe the development of a new high content imaging-based uptake assay using doxorubicin as a fluorescent reporter. Screening the same anti-cancer library of 386 compounds, the new assay identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. Inhibition of MRP1 activity was confirmed using flow cytometry and confocal microscopy-based transport assays. Six drugs (afatinib, celecoxib, doramapimod, mifepristone, MK-2206 and rosiglitazone) were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against vincristine, doxorubicin and etoposide. Mifepristone and doramapimod were most effective in reversal of resistance against vincristine while mifepristone and rosiglitazone were most successful in resensitizing H69AR cells against doxorubicin. Furthermore, resistance towards etoposide was completely reversed in the presence of celecoxib or doramapimod. Selected drugs were also evaluated for resistance reversal in HEK cells that overexpress P-glycoprotein or breast cancer resistance protein. Our results indicate mifepristone and doramapimod as pan inhibitors of these three drug transporters while celecoxib exhibited selective MRP1 inhibition. Together, our findings signify the importance of MRP1 in drug discovery and demonstrate the effectiveness and value of doxorubicin-based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies. On the other hand, anti-cancer drugs that did not interact with MRP1 carry a low risk for developing MRP1-mediated resistance.

Prospective, multicenter study of endovascular repair of aortoiliac and iliac aneurysms using the Gore Iliac Branch Endoprosthesis.

OBJECTIVE: The GORE EXCLUDER Iliac Branch Endoprosthesis (IBE; W. L. Gore and Associates, Flagstaff, Ariz) is an iliac branch stent graft system designed to preserve internal iliac artery perfusion during endovascular repair of aortoiliac aneurysms (AIAs) and common iliac artery (CIA) aneurysms (CIAAs). We report the 6-month primary end point results of the IBE 12-04 United States pivotal trial for endovascular treatment of AIAs and CIAAs using the IBE device. METHODS: The trial prospectively enrolled 63 patients with AIA or CIAA who underwent implantation of the IBE device at 28 centers in the United States from 2013 to 2015. All patients underwent placement of a single IBE device. Twenty-two patients (34.9%) with bilateral CIAs were enrolled after undergoing staged coil or plug embolization (21 of 22) or surgical revascularization (1 of 22) of the contralateral internal iliac artery. Follow-up at 30 days and 6 months included clinical assessment and computed tomography angiography evaluation as assessed by an independent core laboratory. The primary effectiveness end point was freedom from IBE limb occlusion and reintervention for type I or III endoleak and ≥60% stenosis at 6 months, and the secondary effectiveness end point was freedom from new onset of buttock claudication on the IBE side at 6 months. RESULTS: Mean CIA diameter on the IBE side was 41.0 ± 11.4 mm (range, 25.2-76.3 mm). There were no procedural deaths, and technical success, defined as successful deployment and patency of all IBE components and freedom from type I or III endoleak, was 95.2% (60 of 63). Data for 61 patients were available for primary and secondary effectiveness end point analysis. Internal iliac limb patency was 95.1% (58 of 61), and no new type I or III endoleaks or device migrations were observed at 6 months. The three patients with loss of internal iliac limb patency were asymptomatic, and freedom from new-onset buttock claudication on the IBE side was 100% at 6 months. New-onset buttock claudication occurred on the non-IBE treatment side in six of 21 patients (28.6%) who underwent staged internal iliac artery coil embolization. CONCLUSIONS: These results confirm that the IBE device is effective at treating CIAAs and AIAs, maintaining blood flow into the internal iliac artery, and avoiding complications associated with internal iliac artery sacrifice. Follow-up will be continued for 5 years to establish the long-term durability of iliac aneurysm repair with the IBE device.

High-content screening of clinically tested anticancer drugs identifies novel inhibitors of human MRP1 (ABCC1).

Multidrug resistance protein 1 (MRP1/ABCC1), an integral transmembrane efflux transporter, belongs to the ATP-binding cassette (ABC) protein superfamily. MRP1 governs the absorption and disposition of a wide variety of endogenous and xenobiotic substrates including various drugs across organs and physiological barriers. Additionally, its overexpression has been implicated in multidrug resistance in chemotherapy of multiple cancers. Here, we describe the development of a high content imaging-based screening assay for MRP1 activity. This live cell-based automated microscopy assay is very robust and allows simultaneous detection of cell permeable, non-toxic and potent inhibitors. The validity of the assay was demonstrated by profiling a library of 386 anti-cancer compounds, which are under clinical trials, for interactions with MRP1. The assay identified 12 potent inhibitors including two known MRP1 inhibitors, cyclosporine A and rapamycin. On the other hand, MRP1-inhibitory activity of tipifarnib, AZD1208, deforolimus, everolimus, temsirolimus, HS-173, YM201636, ESI-09, TAK-733, and CX-6258 has not been previously reported. Inhibition of MRP1 activity was further validated using flow cytometry and confocal microscopy for the respective detection of calcein and doxorubicin in MRP1-overexpressing cells. Among the identified compounds, tipifarnib, AZD1208, rapamycin, deforolimus, everolimus, TAK-733, and temsirolimus resensitized MRP1-overexpressing H69AR cells towards vincristine, a cytotoxic chemotherapeutic agent, by 2-6-fold. Using purified HEK293 membrane vesicles overexpressing MRP1, MRP2, MRP3, and MRP4, we also demonstrated that the identified compounds exert differential and selective response on the uptake of estradiol glucuronide, an endogenous MRP substrate. In summary, we demonstrated the effectiveness of the high content imaging-based high-throughput assay for profiling compound interaction with MRP1.

Short-term outcomes of the C3 excluder for patients with abdominal aortic aneurysms and unfavorable proximal aortic seal zones.

BACKGROUND: Endovascular abdominal aortic aneurysm repair (EVAR) in patients with unfavorable proximal seal zones remains challenging. The purpose of this study was to identify the incidence of proximal extension cuff usage for type I endoleaks in patients with abdominal aortic aneurysms and unfavorable necks treated with the C3 Excluder repositionable endoprosthesis compared with the traditional Excluder stent-graft. METHODS: This is a retrospective review of patients undergoing EVAR with unfavorable neck anatomy from January 2010 to October 2011 using the Excluder endoprosthesis on the traditional deployment system or the C3 repositionable system. Seventy-seven patients were treated with the Excluder device, with 44 (57%) having unfavorable neck anatomy defined as proximal aortic neck length of <15 mm, neck diameter of >28 mm, neck angulation of >60°, circumferential thrombus of >50% or calcification at the proximal seal zone, or a "reverse taper" on computed tomographic angiography. Of the 44 patients with unfavorable neck anatomy, 24 patients received the C3 Excluder and 20 received the traditional Excluder. RESULTS: The groups' comorbidities, aneurysm characteristics, and high-risk neck criteria were comparable. Initial success was 100% in both groups. Sixteen of the 44 patients (36%) with high-risk neck criteria required proximal extension cuffs for type I endoleaks, with 3 of the 24 patients (13%) in the C3 group compared with 13 of the 20 patients (65%) in the traditional Excluder group requiring proximal extension (P = 0.0005). Operative variables between the two groups were similar. At mean follow-up of 2 months (range: 1-6 months), there were no type I endoleaks or renal artery occlusion, and sac size regression was similar. CONCLUSIONS: The C3 Excluder endoprosthesis significantly reduces the need for proximal extension cuffs in patients with unfavorable aortic neck anatomy compared with the traditional Excluder with identical short-term clinical outcomes. Repositionable grafts could increase the number of patients who can effectively be treated with EVAR.

Endovascular repair of a para-anastomotic pseudoaneurysm after renal autotransplantation: an alternative to open reconstruction.

Renal artery anastomotic pseudoaneurysms are rare after renal transplantation. The etiology tends to be technical, infectious, or degenerative, and repair is difficult with a high postsurgical complication rate. We report the first case of a complex autotransplant renal artery pseudoaneurysm repaired with kissing covered stents. A 52-year-old woman presented with severe left lower quadrant abdominal pain 6 years after a renal autotransplant for ureteral stenosis and recurrent pyelonephritis. A computed tomographic angiography (CTA) scan revealed a bilobed aneurysm arising at the anastomosis between the renal and common iliac arteries. Kissing covered stents were placed within the common iliac artery proximally and extending into the transplant renal artery and external iliac artery. Postdeployment angiography confirmed complete exclusion of the pseudoaneurysm and excellent flow into the transplant kidney and left lower extremity. A follow-up CTA scan at 1 month revealed continued stent-graft patency and complete exclusion of the pseudoaneurysm. An endovascular approach to transplant anastomotic pseduoaneurysms using kissing covered stents is a viable option to exclude aneurysmal changes and preserve flow to the transplanted organ in carefully selected patients.

Creative options for large sheath access during aortic endografting.

Access-related limitations, namely small-caliber vessels and tortuous or calcified stenotic vessels, are often encountered during endovascular aneurysm repair (EVAR) and thoracic EVAR (TEVAR). Overcoming these limitations often requires the creation of a conduit through which the endovascular devices can be delivered. If these limitations are not recognized and respected preoperatively, significant morbidity and mortality may ensue because access-related complications are often addressed in emergent and chaotic situations. There are a variety of conduits described in the literature, each with their own advantages and disadvantages. The present report explores the use of conduits during EVAR and TEVAR by discussing the current literature, and the authors also describe a preferred method to address unfavorable iliac anatomy through the use of endoconduits.

Embolic protection in infrainguinal interventions.

The benefits of the use of embolic protection devices (EPDs) in saphenous vein coronary artery bypass grafts and carotid arteries have been shown, but the utility of their application during infrainguinal endovascular interventions is somewhat unclear. Patients with specific anatomical features or lesion characteristics, or patients undergoing specific types of endovascular interventions may benefit from the off-label use of EPDs, but this has yet to be determined. This report will examine the current literature related to the use of EPDs in infrainguinal endovascular interventions and attempt to identify the patients who would most likely benefit from their use. In addition, the setting in which EPDs have been used at one institution is briefly described. This discussion will serve as a general guideline for the use of EPDs and act as an impetus for the development of future clinical trials to help elucidate the patients who will truly benefit from EPDs use when undergoing lower extremity endovascular revascularization procedures.

Internal endoconduit: an innovative technique to address unfavorable iliac artery anatomy encountered during thoracic endovascular aortic repair.

Various strategies have been used to combat arterial access limitations encountered during thoracic endovascular aortic repair (TEVAR). Most require retroperitoneal dissection or aggressive angioplasty techniques that can lead to devastating complications. We describe a novel technique using an "internal endoconduit." Deployment of an iliac stent graft across the prohibitively stenotic area, followed by angioplasty and controlled rupture of the iliac artery, allows for safe passage of the delivery sheath. Adverse events associated with decreased pelvic perfusion or hemorrhage from iliac artery rupture are theoretically possible but have not been observed. Faced with unfavorable iliac anatomy, we use internal endoconduits rather than retroperitoneal access procedures and believe their use will increase the number of procedures that will be able to be performed through femoral access and substantially reduce the frequency of access-related complications.