Derlin-1 and the E3 Ubiquitin Ligases Hrd1 and gp78 Facilitate Cholera Toxin Retro-translocation.

The endoplasmic reticulum (ER) is the site of folding and assembly for membrane and secretory proteins. When proteins fail to reach their proper conformations, the ER quality control system, ER-associated degradation (ERAD), is responsible for maintaining cell homeostasis via the identification, modification and transport (retro-translocation) of misfolded proteins from the ER to the cytosol for proteasomal degradation. As a model ERAD substrate, Cholera toxin (CT), produced by Vibrio cholerae, relies on its receptor-binding B subunit (CTB) to enter the ER of intestinal epithelial cells, where its catalytic A1 subunit (CTA1) hijacks the ERAD pathway to induce toxicity. This thesis focuses on understanding the role of membrane proteins in the retro-translocation of CTA1. We found that two core components of the retro-translocon, Derlin-1 and the E3 ubiquitin ligases Hrd1 and gp78, facilitate the retro-translocation of CTA1. The expression of dominant negative variants of Derlin-1, Hrd1 and gp78 inhibits the ER-to-cytosol transport of CTA1. Derlin-1, Hrd1 and gp78 interact with CTB and CTA, as well as with the ER oxido-reductase PDI, a lumenal protein known for its role in unfolding the toxin prior to retro-translocation. Furthermore, we reveal a previously unknown role for CTB in the targeting of the toxin to this retro-translocation machinery in the ER membrane. Collectively, these findings suggest a model by which CTB targets the holotoxin to the retro-translocation machinery where PDI unfolds the toxin for retro-translocation through the ER membrane. The function of Hrd1 and gp78 in this model is perplexing, as CTA1 is presumed to be a non-ubiquitinated substrate. However, our data indicate that the catalytic activity of Hrd1, gp78 and an E2 ubiquitin conjugating enzyme dedicated to ERAD are essential for CTA1 transport. Thus, in addition to identifying novel ERAD components involved in CTA1 retro-translocation and elucidating a mechanism by which CT is targeted to the ERAD machinery, we demonstrate that similar to other known ERAD substrates, an intact ubiquitination system is necessary for the ER-to-cytosol transport of CTA1. These data provide important insight into the general mechanism of ERAD and further our understanding of how ERAD substrates utilize the retro-translocation machinery.Ph.D.Cell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64597/1/bernardi_1.pd

The Florida pancreas collaborative next-generation biobank: Infrastructure to reduce disparities and improve survival for a diverse cohort of patients with pancreatic cancer

Background: Well-annotated, high-quality biorepositories provide a valuable platform to support translational research. However, most biorepositories have poor representation of minority groups, limiting the ability to address health disparities. Methods: We describe the establishment of the Florida Pancreas Collaborative (FPC), the first state-wide prospective cohort study and biorepository designed to address the higher burden of pancreatic cancer (PaCa) in African Americans (AA) compared to Non-Hispanic Whites (NHW) and Hispanic/Latinx (H/L). We provide an overview of stakeholders; study eligibility and design; recruitment strategies; standard operating procedures to collect, process, store, and transfer biospecimens, medical images, and data; our cloud-based data management platform; and progress regarding recruitment and biobanking. Results: The FPC consists of multidisciplinary teams from fifteen Florida medical institutions. From March 2019 through August 2020, 350 patients were assessed for eligibility, 323 met inclusion/exclusion criteria, and 305 (94%) enrolled, including 228 NHW, 30 AA, and 47 H/L, with 94%, 100%, and 94% participation rates, respectively. A high percentage of participants have donated blood (87%), pancreatic tumor tissue (41%), computed tomography scans (76%), and questionnaires (62%). Conclusions: This biorepository addresses a critical gap in PaCa research and has potential to advance translational studies intended to minimize disparities and reduce PaCa-related morbidity and mortality

Circulating Cathelicidin Concentrations in a Cohort of Healthy Children: Influence of Age, Body Composition, Gender and Vitamin D Status.

Cathelicidin is an antimicrobial peptide whose circulating levels are related to vitamin D status in adults. This study sought to determine if circulating cathelicidin concentrations in healthy children are related to the age of the child, body composition and vitamin D status at birth and at the time of the study visit. Blood samples were obtained during yearly visits from 133 children, ages 2-7, whose mothers had participated in a pregnancy vitamin D supplementation RCT. Radioimmunoassay and ELISA were performed to analyze 25(OH)D and cathelicidin, respectively. Statistical analyses compared cathelicidin concentrations with concentrations of 25(OH)D at various time points (maternal levels throughout pregnancy, at birth, and child's current level); and with race/ethnicity, age, gender, BMI, percent fat, and frequency of infections using Student's t-test, χ2, Wilcoxon ranked-sum analysis, and multivariate regression. The cohort's median cathelicidin concentration was 28.1 ng/mL (range: 5.6-3368.6) and did not correlate with 25(OH)D, but was positively correlated with advancing age (ρ = 0.236 & p = 0.005, respectively). Forty patients evaluated at two visits showed an increase of 24.0 ng/mL in cathelicidin from the first visit to the next (p<0.0001). Increased age and male gender were correlated with increased cathelicidin when controlling for race/ethnicity, percent fat, and child's current 25(OH)D concentration (p = 0.028 & p = 0.047, respectively). This study demonstrated that as children age, the concentration of cathelicidin increases. Furthermore, male gender was significantly associated with increased cathelicidin concentrations. The lack of association between vitamin D status and cathelicidin in this study may be due to the narrow range in observed 25(OH)D values and warrants additional studies for further observation

Multivariable Linear Regression Models Assessing Factors Associated with Natural Logarithm for Cathelicidin.

<p>Multivariable Linear Regression Models Assessing Factors Associated with Natural Logarithm for Cathelicidin.</p

Child Sociodemographics and Clinical Characteristics.

<p>Child Sociodemographics and Clinical Characteristics.</p

Cathelicidin Concentration by Age.

<p>There was a significant positive correlation between age and cathelicidin concentration (ρ = 0.236, p = 0.005); ρ value represents Spearman correlation. The upper and lower edges of the box represent the 75^th percentile and the 25^th percentile of cathelicidin concentration, respectively, measured by ELISA and reported in ng/mL.</p

Bivariate Analysis between Cathelicidin and Categorical Variables.

<p>Bivariate Analysis between Cathelicidin and Categorical Variables.</p

Change of Cathelicidin Concentration over Time.

<p>In a subset of 40 children with two consecutive visits roughly twelve months apart, the median increase in cathelicidin concentration from the first visit to the second visit was 24.0 ng/mL (ranging from -1.9 ng/mL to +223.4ng/mL, p≤0.0001); ρ value represents Spearman correlation. The upper and lower edges of the box represent the 75^th percentile and the 25^th percentile of cathelicidin concentration, respectively, reported in ng/mL.</p

The Florida Pancreas Collaborative Next-Generation Biobank: Infrastructure to Reduce Disparities and Improve Survival for a Diverse Cohort of Patients with Pancreatic Cancer

Background: Well-annotated, high-quality biorepositories provide a valuable platform to support translational research. However, most biorepositories have poor representation of minority groups, limiting the ability to address health disparities. Methods: We describe the establishment of the Florida Pancreas Collaborative (FPC), the first state-wide prospective cohort study and biorepository designed to address the higher burden of pancreatic cancer (PaCa) in African Americans (AA) compared to Non-Hispanic Whites (NHW) and Hispanic/Latinx (H/L). We provide an overview of stakeholders; study eligibility and design; recruitment strategies; standard operating procedures to collect, process, store, and transfer biospecimens, medical images, and data; our cloud-based data management platform; and progress regarding recruitment and biobanking. Results: The FPC consists of multidisciplinary teams from fifteen Florida medical institutions. From March 2019 through August 2020, 350 patients were assessed for eligibility, 323 met inclusion/exclusion criteria, and 305 (94%) enrolled, including 228 NHW, 30 AA, and 47 H/L, with 94%, 100%, and 94% participation rates, respectively. A high percentage of participants have donated blood (87%), pancreatic tumor tissue (41%), computed tomography scans (76%), and questionnaires (62%). Conclusions: This biorepository addresses a critical gap in PaCa research and has potential to advance translational studies intended to minimize disparities and reduce PaCa-related morbidity and mortality