Systems-Based Design of Bi-Ligand Inhibitors of Oxidoreductases: Filling the Chemical Proteomic Toolbox

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Loss of the extraproteasomal ubiquitin receptor Rings lost impairs ring canal growth in Drosophila oogenesis

Rings lost fulfills a novel function in Drosophila development for a ubiquitin receptor as an essential mediator of ring canal growth during oogenesis

The ubiquitin-associated (UBA) 1 domain of Schizosaccharomyces pombe Rhp23 is essential for the recognition of ubiquitin-proteasome system substrates both in vitro and in vivo

The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys(48)-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2

Structural Analysis of the UBA Domain of X-linked Inhibitor of Apoptosis Protein Reveals Different Surfaces for Ubiquitin-Binding and Self-Association

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) belong to a pivotal antiapoptotic protein family that plays a crucial role in tumorigenesis, cancer progression, chemoresistance and poor patient-survival. X-linked inhibitor of apoptosis protein (XIAP) is a prominent member of IAPs attracting intense research because it has been demonstrated to be a physiological inhibitor of caspases and apoptosis. Recently, an evolutionarily conserved ubiquitin-associated (UBA) domain was identified in XIAP and a number of RING domain-bearing IAPs. This has placed the IAPs in the group of ubiquitin binding proteins. Here, we explore the three-dimensional structure of the XIAP UBA domain (XIAP-UBA) and how it interacts with mono-ubiquitin and diubiquitin conjugates. PRINCIPAL FINDINGS: The solution structure of the XIAP-UBA domain was determined by NMR spectroscopy. XIAP-UBA adopts a typical UBA domain fold of three tightly packed alpha-helices but with an additional N-terminal 3(10) helix. The XIAP-UBA binds mono-ubiquitin as well as Lys48-linked and linear-linked diubiquitins at low-micromolar affinities. NMR analysis of the XIAP-UBA-ubiquitin interaction reveals that it involves the classical hydrophobic patches surrounding Ile44 of ubiquitin and the conserved MGF/LV motif surfaces on XIAP-UBA. Furthermore, dimerization of XIAP-UBA was observed. Mapping of the self-association surface of XIAP-UBA reveals that the dimerization interface is formed by residues in the N-terminal 3(10) helix, helix alpha1 and helix alpha2, separate from the ubiquitin-binding surface. CONCLUSION: Our results provide the first structural information of XIAP-UBA and map its interaction with mono-ubiquitin, Lys48-linked and linear-linked diubiquitins. The notion that XIAP-UBA uses different surfaces for ubiquitin-binding and self-association provides a plausible model to explain the reported selectivity of XIAP in binding polyubiquitin chains with different linkages.published_or_final_versio

Hard Labor: Pursuing Economic Citizenship and Legal Recognition of Certified Professional Midwives in Alabama

Until 1976, women in Alabama could choose to make use of a midwife when they gave birth. In that year, the Alabama state legislature outlawed the practice. This dissertation explores the consequences of that decision as well as the efforts of contemporary non-nurse midwives, also known as Certified Professional Midwives (CPM’s), to re-establish the practice as an option available to birthing women in the state. In order to address the consequences of outlawing non-nurse midwives in the state of Alabama a mixed methodology approach is applied. Two years of ethnographic data collection approached with a feminist and cultural anthropology lens, reveal that the lack of medical infrastructure within the state of Alabama prohibits the ability for CPM’s to practice safely. This is owed to historically grounded stigma in racism and classism. As a result, the current CPM community within the state of Alabama, along with their clientele, is predominantly white. This is reflected in the case studies within the dissertation as all the families and care providers, regardless of clinical expertise, are all white. An examination of cesarean rates via quantitative analysis supports the historical and ethnographic findings. Cesarean rates are highest within counties that have a low median household income, and a population that is predominately African American. The dissertation features five case studies of women who gave birth attended by a CPM. By relating the experiences of the birthing mothers, a CPM, and certified medical professionals, the dissertation offers evidence of the kind of supplemental medical care and knowledge that can be offered by practitioners of midwifery. At the same time, while contemporary midwives such as the one featured here offer important medical service to their clients, they are not equipped to or knowledgeable about political work necessary to push for the re-legalization of midwifery. This dissertation thus sheds light on the challenges facing midwives who would prefer to work openly and legally in the state. Ultimately what is revealed is the value of supplementary healthcare networks within the state. While care and birth services provided by CPM’s is not readily accessible to all, those giving birth in Alabama can find support within the current system through supplementary healthcare networks. These networks include doulas, lactation support groups, babywearing groups, etc. It is a piecemeal system to be sure, but it is a piecemeal system that is working diligently to unlearn biases, and support women and birthing families. However, it is important to understand that the supplemental networks cannot fully address the larger structural crisis that is a lack of infrastructure within the state’s medical system. Ideally, a system that utilizes Obstetricians, Nurse Midwives, and Non-nurse Midwives, all with mutual respect for their own expertise, would exist to provide quality care to women throughout the state

Red Hills to Gulf Shores: Autographics

Reception for exhibitio

Katrina, One Year Later: Three Perspectives

This collection of three photographic essays documenting the 2006 Mississippi Gulf Coast offers multiple perspectives on Hurricane Katrina's aftermath. Bruce West, Todd Bertolaet, and David Wharton traveled to Bay St. Louis, Gulfport, Long Beach, Pass Christian, Biloxi, Waveland, and points in between uncovering the new post-Hurricane Katrina environment