Showing papers by "Lance Wells published in 2012"

Structural reorganization of the interleukin-7 signaling complex.

Abstract: We report here an unliganded receptor structure in the common gamma-chain (γc) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 A resolution reveals a homodimer forming an “X” geometry looking down onto the cell surface with the C termini of the two chains separated by 110 A and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γc/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4+ T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα–γc heterodimer to the active IL-7–IL-7Rα–γc ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γc from a distance of 110 A to less than 30 A at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7– and γc-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γc receptors that form inactive homodimers and heterodimers independent of their cytokines.

Human UDP-α-d-xylose Synthase and Escherichia coli ArnA Conserve a Conformational Shunt That Controls Whether Xylose or 4-Keto-Xylose Is Produced

Abstract: Proteoglycans act as receptors for growth factors and are essential for cell proliferation, migration and adhesion.1–3 Disrupting proteoglycan biosynthesis can attenuate tumor growth and progression; thus controlling proteoglycan biosynthesis is a promising strategy for treating cancer.2–5 The biosynthesis of most proteoglycans begins with the covalent attachment of xylose to the hydroxyl of a serine.6–8 In mammals, xylose is also used in non-mucin O-glycosylation of key regulatory proteins such as Notch and α-dystroglycan that play important roles in cancer progression and metastasis.9–12 The nucleotide sugar donor that initiates xylose transfer is produced by UDP-α-d-xylose synthase (UXS; E.C. 4.1.1.35), a member of the short-chain dehydrogenase/reductase (SDR) family. Understanding the mechanism of UXS may contribute to the design of strategies that will slow or prevent metastasis. SDR enzymes share a common catalytic domain containing a Rossmann fold for binding the NAD+ cofactor.13 UXS is further classified as an ‘extended’ SDR due to an inserted nucleotide sugar binding domain (NSBD).14,15 The extended SDR subfamily includes nucleotide sugar epimerases, dehydratases and decarboxylases, all of which use a similar NAD+-dependent mechanism. The recent crystal structure of human UXS (hUXS) in complex with NAD+ and UDP has shed light on the mechanism (Figure 1).15 Briefly, hUXS uses a bound NAD+ cofactor to oxidize of the C4′ atom of UDP-α-d-glucuronic acid (UGA) to produce UDP-α-d-4-keto-glucuronic acid. The unstable β-keto-acid intermediate decarboxylates to form the more stable UDP-α-d-4-keto-xylose (UX4O). Finally, hUXS uses the NADH cofactor produced in the first step to reduce the UX4O intermediate to UDP-α-d-xylose (UDX). hUXS purifies with a bound cofactor and therefore has no requirement for additional NAD+.16 Despite this observation, several reports have demonstrated that adding exogenous NAD+ can stimulate UXS activity by 10–104%.15,17–21 This stimulation has been attributed to a significant contamination of apoenzyme in purified UXS.15,17 Figure 1 UXS and ArnA have similar catalytic mechanisms. UDP-glucuronic acid (UGA) decarboxylases catalyze NAD+-dependent oxidation of the C4′ position of substrate to form UDP-4-keto-xylose (UX4O) and NADH. ArnA releases UX4O and NADH as products. UXS ... We present evidence that the stimulatory effect of exogenous NAD+ is due to the accumulation of apo-hUXS during catalytic turnover. We show that the hUXS mechanism shunts UGA into two different paths: (i) the slow production of NADH and UX4O, or (ii) the preferred conversion to UDX. The first path results in an inactive apoenzyme that can be rescued by exogenous NAD+. In contrast to hUXS, the homologous UGA decarboxylase domain of Escherichia coli ArnA uses NAD+ as a cosubstrate and releases NADH and UX4O as products (Figure 1).22,23 We show that cofactor binding in UXS and ArnA invokes a conserved, cooperative conformational change. Interestingly, ArnA conserves the bifurcated mechanism of hUXS and can produce UDX, albeit rather inefficiently. Using a new crystal structure of hUXS, we identify the flexible active site elements that contribute to the UX4O and NADH shunt. We present a model for this bifurcated mechanism that explains the stimulatory effect of exogenous NAD+ on hUXS and the production of UDX by ArnA.

Program and abstracts for the 2012 Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology

Abstract: Time 8:30 Glycobiology of Human Pluripotent Stem Cells; Steve Dalton, University of Georgia #1 9:00 Siglec-Sialoglycan Binding Regulates Cell-Cell Interactions; Ronald Schnaar, Johns Hopkins School of Medicine 9:30 Stem Cell Therapies in Epidermolysis Bullosa; Angela Christiano, Columbia University Medical Center 10:00 – 10:30 am Coffee Break (Grande Foyer and Grande Ballroom A) 10:30 am – 12:00 pm Plenary II: Biomaterials and Matrix Engineering (Grande Ballroom B & C) Chair: Adam Engler, University of California, San Diego 10:30 Hydrogels as Synthetic Extracellular Matrices; Kristi Anseth, University of Colorado at Boulder 11:00 Mechanical Regulation of Cell Adhesion and Function; Christopher Chen, University of Pennsylvania Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology Conference Program by A rm en Peosyan on N ovem er 2, 2012 http://glycfordjournals.org/ D ow nladed from