Identifying substrate proteins for GAN1 and Keap1

Abstract only availableGAN1 and Keap1 are proteins characterized by a N-terminal BTB domain and a C-terminal Kelch repeat domain. Both of these domains are protein-protein interaction domains, suggesting that these BTB-Kelch proteins form signaling complexes in cells. Previous work has suggested that BTB-Kelch proteins function as substrate adaptor proteins for Cul3-dependent E3 ubiquitin ligase complexes. The goal of this project was to identify substrate proteins of GAN1 and Keap1. This information will be particularly useful when for understanding Giant Axonal Neuropathy, a sensorimotor disease characterized by excessive accumulation of neurofilaments in neurons that contain mutated GAN1 genes. We used an affinity purification approach to identify candidate substrate proteins for GAN1 and Keap1. Recombinant GAN1 and Keap1 genes containing a C-terminal chitin binding domain (CBD) were inserted into pBabe puro vectors. These vectors were used to generate virus stocks, which were used to infect a microglial cell line, BV-2. Stable cell lines were generated using puromycin selection. A mock-infected cell line was generated in parallel. When the cell lines were confluent, the cells were lysed using a 0.1% SDS RIPA solution and chitin beads were used to precipitate the CBD-tagged proteins. Western blot analyses were performed to determine if the purification of the CBD-tagged proteins was successful. No CBD-tagged proteins were identified in our first pull-down experiment. We are currently reexamining the precipitation protocol and preparing to lysate the same set of cells.Life Sciences Undergraduate Research Opportunity Progra

Nuclear Localization of Ikappa Balpha Is Mediated by the Second Ankyrin Repeat: the Ikappa Balpha Ankyrin Repeats Define a Novel Class of cis-Acting Nuclear Import Sequences

The ability of the Ikappa Balpha protein to sequester dimeric NF-kappa B/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-kappa B/Rel family members. Ikappa Balpha can also act in the nucleus as a postinduction repressor of NF-kappa B/Rel proteins. The mechanism by which Ikappa Balpha enters the nucleus is not known, as Ikappa Balpha lacks a discernible classical nuclear localization sequence (NLS). We now report that nuclear localization of Ikappa Balpha is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of Ikappa Balpha . Furthermore, a region encompassing the second ankyrin repeat of Ikappa Balpha is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in Ikappa Balpha suggests that cytoplasmic sequestration of the NF-kappa B/Rel-Ikappa Balpha complex is a consequence of the mutual masking of the NLS within NF-kappa B/Rel proteins and the import sequence within Ikappa Balpha . Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), as the ARDs from two other ARD-containing proteins, 53BP2 and GABPbeta , are also able to function as nuclear import sequences. We propose that the Ikappa Balpha ankyrin repeats define a novel class of cis-acting nuclear import sequences

Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress

Sensitization of neuroblastoma to apoptosis mediated by oncogene-specific antisense peptide nucleic acid oligomers [abstract]

Abstract only availableFaculty Mentor: Michael R. Lewis, Ph.D., Veterinary Medicine and Surgery/Radiology/NuclearApoptosis, or programmed cell death, is a major pathway by which chemotherapeutic drugs kill tumor cells. The B-cell lymphoma/leukemia-2 (bcl-2) and FLICE-inhibitory (FLIP) cellular oncogenes promote tumor cell survival, by blocking apoptotic signals or mechanisms of action. The objective of the present studies was to determine whether antisense peptide nucleic acid (PNA) oligomers targeted to bcl-2 and FLIP sensitize resistant neuroblastoma cells to Fas-mediated apoptosis. The Fas receptor pathway has been shown to be an important mediator of drug-induced apoptosis, and the development of drug resistance has been linked to deficient activation of Fas. IMR-32 neuroblastoma cells overexpress the oncoprotein bcl-2, which inhibits mitochondrial activation of apoptosis. These cells also overexpress the caspase-8 inhibitor FLIP, which blocks poly(ADP-ribose) polymerase cleavage and apoptotic DNA degradation. SH-SY5Y neuroblastoma cells express lower levels of bcl-2 protein and have minimal caspase-8 expression. Using lipofectamine PLUS as a delivery vehicle, the two cell lines were treated with anti-bcl-2 or anti-FLIP PNAs, a combination of the two, or a negative control PNA not complementary to any known mammalian DNA or RNA sequence. SH-SY5Y cells showed no decreases in bcl-2 or FLIP protein expression following antisense PNA treatment. In contrast, IMR-32 cells exhibited a dose-dependent 40% decrease in bcl-2 expression following incubation with anti-bcl-2 PNA, but no antisense effect on FLIP expression. After PNA treatment, IMR-32 and SH-SY5Y cells were incubated with anti-Fas monoclonal antibody CH11. Cell death was measured by the MTT colorimetric assay, and apoptosis was evaluated by the TUNEL method. The results of these studies allowed assessment of differential sensitization of these two neuroblastoma cell lines to Fas-mediated apoptosis induced by antisense PNAs

Severity of oEsophageal Anastomotic Leak in patients after oesophagectomy:the SEAL score

Background Anastomotic leak (AL) is a common but severe complication after oesophagectomy. It is unknown how to determine the severity of AL objectively at diagnosis. Determining leak severity may guide treatment decisions and improve future research. This study aimed to identify leak-related prognostic factors for mortality, and to develop a Severity of oEsophageal Anastomotic Leak (SEAL) score. Methods This international, retrospective cohort study in 71 centres worldwide included patients with AL after oesophagectomy between 2011 and 2019. The primary endpoint was 90-day mortality. Leak-related prognostic factors were identified after adjusting for confounders and were included in multivariable logistic regression to develop the SEAL score. Four classes of leak severity (mild, moderate, severe, and critical) were defined based on the risk of 90-day mortality, and the score was validated internally. Results Some 1509 patients with AL were included and the 90-day mortality rate was 11.7 per cent. Twelve leak-related prognostic factors were included in the SEAL score. The score showed good calibration and discrimination (c-index 0.77, 95 per cent c.i. 0.73 to 0.81). Higher classes of leak severity graded by the SEAL score were associated with a significant increase in duration of ICU stay, healing time, Comprehensive Complication Index score, and Esophagectomy Complications Consensus Group classification. Conclusion The SEAL score grades leak severity into four classes by combining 12 leak-related predictors and can be used to the assess severity of AL after oesophagectomy.The Severity of oEsophageal Anastomotic Leak (SEAL) score was developed using data from the TENTACLE-Esophagus study, an international, multicentre retrospective cohort study including 1509 patients with anastomotic leak after oesophagectomy. The SEAL score was developed to determine anastomotic leak severity at diagnosis, and combines 12 leak-related parameters at diagnosis. The score may be useful in clinical practice and could improve future research.</p

Treatment of anastomotic leak after oesophagectomy for oesophageal cancer:large, collaborative, observational TENTACLE cohort study

Background: Anastomotic leak is a severe complication after oesophagectomy. Anastomotic leak has diverse clinical manifestations and the optimal treatment strategy is unknown. The aim of this study was to assess the efficacy of treatment strategies for different manifestations of anastomotic leak after oesophagectomy. Methods: A retrospective cohort study was performed in 71 centres worldwide and included patients with anastomotic leak after oesophagectomy (2011-2019). Different primary treatment strategies were compared for three different anastomotic leak manifestations: interventional versus supportive-only treatment for local manifestations (that is no intrathoracic collections; well perfused conduit); drainage and defect closure versus drainage only for intrathoracic manifestations; and oesophageal diversion versus continuity-preserving treatment for conduit ischaemia/necrosis. The primary outcome was 90-day mortality. Propensity score matching was performed to adjust for confounders. Results: Of 1508 patients with anastomotic leak, 28.2 per cent (425 patients) had local manifestations, 36.3 per cent (548 patients) had intrathoracic manifestations, 9.6 per cent (145 patients) had conduit ischaemia/necrosis, 17.5 per cent (264 patients) were allocated after multiple imputation, and 8.4 per cent (126 patients) were excluded. After propensity score matching, no statistically significant differences in 90-day mortality were found regarding interventional versus supportive-only treatment for local manifestations (risk difference 3.2 per cent, 95 per cent c.i. -1.8 to 8.2 per cent), drainage and defect closure versus drainage only for intrathoracic manifestations (risk difference 5.8 per cent, 95 per cent c.i. -1.2 to 12.8 per cent), and oesophageal diversion versus continuity-preserving treatment for conduit ischaemia/necrosis (risk difference 0.1 per cent, 95 per cent c.i. -21.4 to 1.6 per cent). In general, less morbidity was found after less extensive primary treatment strategies. Conclusion: Less extensive primary treatment of anastomotic leak was associated with less morbidity. A less extensive primary treatment approach may potentially be considered for anastomotic leak. Future studies are needed to confirm current findings and guide optimal treatment of anastomotic leak after oesophagectomy.</p

Intrathoracic vs Cervical Anastomosis After Totally or Hybrid Minimally Invasive Esophagectomy for Esophageal Cancer A Randomized Clinical Trial

Background: Transthoracic minimally invasive esophagectomy (MIE) is increasingly performed as part of curative multimodality treatment. There appears to be no robust evidence on the preferred location of the anastomosis after transthoracic MIE. Objective: To compare an intrathoracic with a cervical anastomosis in a randomized clinical trial. Design, Setting, and Participants: This open, multicenter randomized clinical superiority trial was performed at 9 Dutch high-volume hospitals. Patients with midesophageal to distal esophageal or gastroesophageal junction cancer planned for curative resection were included. Data collection occurred from April 2016 through February 2020. Intervention: Patients were randomly assigned (1:1) to transthoracic MIE with intrathoracic or cervical anastomosis. Main Outcomes and Measures: The primary end point was anastomotic leakage requiring endoscopic, radiologic, or surgical intervention. Secondary outcomes were overall anastomotic leak rate, other postoperative complications, length of stay, mortality, and quality of life. Results: Two hundred sixty-two patients were randomized, and 245 were eligible for analysis. Anastomotic leakage necessitating reintervention occurred in 15 of 122 patients with intrathoracic anastomosis (12.3%) and in 39 of 123 patients with cervical anastomosis (31.7%; risk difference, -19.4% [95% CI, -29.5% to -9.3%]). Overall anastomotic leak rate was 12.3% in the intrathoracic anastomosis group and 34.1% in the cervical anastomosis group (risk difference, -21.9% [95% CI, -32.1% to -11.6%]). Intensive care unit length of stay, mortality rates, and overall quality of life were comparable between groups, but intrathoracic anastomosis was associated with fewer severe complications (risk difference, -11.3% [-20.4% to -2.2%]), lower incidence of recurrent laryngeal nerve palsy (risk difference, -7.3% [95% CI, -12.1% to -2.5%]), and better quality of life in 3 subdomains (mean differences: dysphagia, -12.2 [95% CI, -19.6 to -4.7]; problems of choking when swallowing, -10.3 [95% CI, -16.4 to 4.2]; trouble with talking, -15.3 [95% CI, -22.9 to -7.7]). Conclusions and Relevance: In this randomized clinical trial, intrathoracic anastomosis resulted in better outcome for patients treated with transthoracic MIE for midesophageal to distal esophageal or gastroesophageal junction cancer. Trial Registration: Trialregister.nl Identifier: NL4183 (NTR4333)

Fiber Type-Specific Nitric Oxide Protects Oxidative Myofibers against Cachectic Stimuli

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia