Inherent properties of adenylosuccinate lyase could explain S-Ado/SAICAr ratio due to homozygous R426H and R303C mutations.

Adenylosuccinate lyase (ADSL) is a homotetrameric enzyme involved in the de novo purine biosynthesis pathway and purine nucleotide cycle. Missense mutations in the protein lead to ADSL deficiency, an inborn error of purine metabolism characterized by neurological and physiological symptoms. ADSL deficiency is biochemically diagnosed by elevated levels of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado), the dephosphorylated derivatives of the substrates. S-Ado/SAICAr ratios have been associated with three phenotypic groups. Different hypotheses to explain these ratios have been proposed. Recent studies have focused on measuring activity on the substrates independently. However, it is important to examine mixtures of the substrates to determine if mutations affect enzyme activity on both substrates similarly in these conditions. The two substrates may experience an indirect communication due to being acted upon by the same enzyme, altering their activities from the non-competitive case. In this study, we investigate this hidden coupling between the two substrates. We chose two mutations that represent extremes of the phenotype, R426H and R303C. We describe a novel electrochemical-detection method of measuring the kinetic activity of ADSL in solution with its two substrates at varying concentration ratios. Furthermore, we develop an enzyme kinetic model to predict substrate activity from a given ratio of substrate concentrations. Our findings indicate a non-linear dependence of the activities on the substrate ratios due to competitive binding, distinct differences in the behaviors of the different mutations, and S-Ado/SAICAr ratios in patients could be explained by inherent properties of the mutant enzyme.

Structural and biochemical characterization of human adenylosuccinate lyase (ADSL) and the R303C ADSL deficiency-associated mutation.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. ADSL executes two nonsequential steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide, which occurs in the de novo synthesis of IMP, and the conversion of adenylosuccinate to AMP, which occurs in the de novo synthesis of AMP and also in the purine nucleotide cycle, using the same active site. Mutation of ADSL's arginine 303 to a cysteine is known to lead to ADSL deficiency. Interestingly, unlike other mutations leading to ADSL deficiency, the R303C mutation has been suggested to more significantly affect the enzyme's ability to catalyze the conversion of succinyladenosine monophosphate than that of SAICAR to their respective products. To better understand the causation of disease due to the R303C mutation, as well as to gain insights into why the R303C mutation potentially has a disproportional decrease in activity toward its substrates, the wild type (WT) and the R303C mutant of ADSL were investigated enzymatically and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL's cooperativity. By utilizing this information, a model for the interaction between ADSL and SAICAR is proposed.

Adipose Derived Cells and Tissues for Regenerative Medicine.

Adipose tissue is now recognized as a complex organ serving endocrine, immune, and metabolic functions. Adipose depots are composed of mature adipocytes as well as stromal vascular fraction (SVF) cells, a heterogeneous population of B and T lymphocytes, endothelial cells, macrophages, pericytes, smooth muscle, and stromal cells that can be isolated by enzymatic digestion. When placed into culture medium, a subset of the SVF cells can adhere to the plastic surface and expand in number. This latter population, known as "adipose-derived stromal/stem cells" (ASC), exhibits trilineage (adipo-, chondro-, osteo-) differentiation potential. There are currently more than 180 clinical studies underway worldwide exploring the regenerative medical application of SVF cells and ASC in a range of medical conditions. Plastic surgeons have a particular interest in the use of autologous fat and SVF enhanced fat for cosmetic and reconstructive surgical procedures. Orthopedic and craniofacial surgeons have begun to use ASC to treat bone and musculoskeletal defects with success. Furthermore, studies are underway to exploit the immunomodulatory function of ASC to treat immune-mediated disorders such as Crohn's disease. Indeed, it is postulated that adipose tissue and cells modulate tissue regeneration and inflammatory responses through their secretion of paracrine factors. Continued advances in this emerging field will require harmonization of international standards and guidelines defining the release criteria as well as the safety and efficacy of adipose-derived cells and tissues. Currently, there are no accepted standard guidelines for autologous fat harvesting technique, processing, or method of injection. Close collaboration between academia, industry, and regulatory authorities will be necessary to ensure that adipose-derived products are affordable and quality controlled throughout the globe.