The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression.

Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors.

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (>1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (K(i) = 6-7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).

Identification and characterization of a novel and functional murine Pin1 isoform.

Pin1, a phosphorylation-dependent peptidyl-prolyl cis/trans isomerase (PPIase), regulates the activity of a number of cell cycle regulators, transcription factors, and microtubule-associated tau. Aberrant expression of Pin1 is implicated in carcinogenesis and neurodegenerative diseases. Yet, there are discrepancies regarding its biological significance in different organisms. Pin1 was essential in HeLa cells, while Pin1-deficient mice showed no lethal phenotypes. We here identified a novel murine Pin1 isoform (mPin1L) consisting of the WW domain and the PPIase domain. Murine Pin1L shares 92% sequence identity with the wild-type Pin1 and shows wide tissue distribution with highest levels in mouse testis. The recombinant mPin1L is enzymatically active, but is approximately three times less efficient than Pin1 in catalyzing the cis/trans isomerization. These data suggest that mPin1L may serve as a surrogate for Pin1. The finding provides insights into phenotypic consequences for Pin1-null mice and may facilitate future biological study and pharmacological development in mice.

Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1.

Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming.

Formation of atypical isoaspartyl (isoAsp) sites in peptides and proteins via the deamidation-linked isomerization of asparaginyl-Xaa bonds or direct isomerization of aspartyl-Xaa bonds is a major contributor to spontaneous protein damage under mild conditions. This nonenzymatic reaction reroutes the Asx-Xaa peptide bond through the beta-carbonyl of asparaginyl or aspartyl residues, thereby adding an extra carbon to the polypeptide backbone. Formation of isoAsp has been implicated in protein inactivation, aggregation, degradation, and autoimmunity. Knowing the location of isoAsp sites in proteins is important for understanding mechanisms of protein damage and for characterizing protein pharmaceuticals. Here we present a simple nonradioactive method for direct localization of isoAsp residues in peptides or proteins. Using three model peptides, we demonstrate that isoAsp linkages can be cleaved selectively and in high yield by a two-step process in which (i) the isoAsp linkage is converted into a succinimide on incubation with S-adenosyl-l-methionine and the commercially available enzyme, protein l-isoaspartyl-O-methyltransferase, and (ii) the succinimidyl bond is then cleaved by hydroxylamine under conditions that minimize cleavage of the traditional hydroxylamine-sensitive Asn-Gly and related peptide bonds. Location of the isoAsp linkage is then inferred by identifying the cleavage products by mass spectrometry or N-terminal sequencing.

Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain.

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.