Studies on the structure and mechanisms of methylmalonyl-CoA mutase

SIGLEAvailable from British Library Document Supply Centre- DSC:D062651 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility

The N-terminus of the Breast Cancer-1 predisposition protein (BRCA1) associates with the BRCA1-associated RING domain-1 protein (BARD1) to form a heterodimer, which exhibits ubiquitin ligase activity that is abrogated by known cancer-associated BRCA1 missense mutations. The majority of missense substitutions identified in patients with a personal or a family history of disease have not been followed in pedigrees, nor there is a functional understanding of their impact. We have examined, by extensive missense substitution, the interaction of BRCA1 with components that contribute to its ubiquitin ligase activity, BARD1 and the E2 ubiquitin-conjugating enzyme, UbcH5a. Selection from a randomly generated library of BRCA1 missense mutations for variants that inhibit the interaction with these components identified substitutions in residues found altered in patient DNA, indicating a correlation between loss of component-binding and propensity to disease development. We further show that the BRCA1:E2 interaction is sensitive to substitutions in all structural elements of the BRCA1 N-terminus, whereas the BARD1 interaction is sensitive to a subset of BRCA1 substitutions, which also inhibit E2-binding. Patient variants that inhibit the BRCA1:E2 interaction show loss of ubiquitin ligase activity and correlate with disease susceptibility and theoretical predictions of pathogenicity. These data link the loss of ubiquitin ligase activity, through loss of E2-binding, to the majority of non-polymorphic patient variants described within the N-terminus of BRCA1 and illustrate the likely significant role of BRCA1 ubiquitin ligase activity in tumour suppression

Crystal structure of R120G disease mutant of human αB-Crystallin Domain Dimer shows closure of a groove

Small heat shock proteins (sHsps) form large cytosolic assemblies from an “α-crystallin domain” (ACD) flanked by sequence extensions. Mutation of a conserved arginine in the ACD of several human sHsp family members causes many common inherited diseases of the lens and neuromuscular system. The mutation R120G in αB crystallin causes myopathy, cardiomyopathy and cataract. We have solved the x-ray structure of the excised ACD dimer of human αB R120G close to physiological pH and compared it with several recently determined wild-type vertebrate ACD dimer structures. Wild-type excised ACD dimers have a deep groove at the interface floored by a flat extended “bottom sheet”. Solid-state NMR studies of large assemblies of full-length αB-crystallin have shown that the groove is blocked in the ACD dimer by curvature of the “bottom sheet”. The crystal structure of R120G ACD dimer also reveals a closed groove, but here the “bottom sheet” is flat. Loss of Arg120 results in rearrangement of an extensive array of charged interactions across this interface. His83 and Asp80 on movable arches either side of the interface close the groove by forming two new salt bridges. The residues involved in this extended set of ionic interactions are conserved in Hsp27, Hsp20, αA and αB-crystallin sequences. They are not conserved in Hsp22, where mutation of the equivalent of Arg120 causes neuropathy. We speculate that the αB R120G mutation disturbs oligomer dynamics causing the growth of large soluble oligomers that are toxic to cells by blocking essential processes

Utrophin Binds Laterally along Actin Filaments and Can Couple Costameric Actin with Sarcolemma When Overexpressed in Dystrophin-deficient Muscle

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments