Differences in the dynamics of the tandem‐SH2 modules of the Syk and ZAP‐70 tyrosine kinases

The catalytic activity of Syk‐family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine‐based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP‐70, that its conformation more closely resembles that of the peptide‐bound state, rather than the autoinhibited state. Hydrogen–deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP‐70 differ, with most of these differences occurring in the C‐terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP‐70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP‐70, consistent with tighter control of downstream signaling pathways in T cells.     

Organization of Enzyme Concentration across the Metabolic Network in Cancer Cells

Rapid advances in mass spectrometry have allowed for estimates of absolute concentrations across entire proteomes, permitting the interrogation of many important biological questions. Here, we focus on a quantitative aspect of human cancer cell metabolism that has been limited by a paucity of available data on the abundance of metabolic enzymes. We integrate data from recent measurements of absolute protein concentration to analyze the statistics of protein abundance across the human metabolic network. At a global level, we find that the enzymes in glycolysis comprise approximately half of the total amount of metabolic proteins and can constitute up to 10% of the entire proteome. We then use this analysis to investigate several outstanding problems in cancer metabolism, including the diversion of glycolytic flux for biosynthesis, the relative contribution of nitrogen assimilating pathways, and the origin of cellular redox potential. We find many consistencies with current models, identify several inconsistencies, and find generalities that extend beyond current understanding. Together our results demonstrate that a relatively simple analysis of the abundance of metabolic enzymes was able to reveal many insights into the organization of the human cancer cell metabolic network.     

Dynamics and memory of heterochromatin in living cells

Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.     

Frequency of polymorphisms for alleles encoding for liver proteins of domesticated mice

Three different inbred strains of mice have been crossed with a lethal albino line (cch/c3H) and the liver polypeptides of the parents and offspring examined by two-dimensional polyacrylamide gel electrophoresis for evidences of protein polymorphisms, different alleles of which have gone to fixation in different strains. In the battery of polypeptides considered most favorable for scoring, 3.3 +/- 1.6 percent of the battery exhibited paired variants and 1.6 +/- 1.2 percent, unpaired. An adjustment for the fact the same allele of a biallelic polymorphism may go to fixation in two inbred lines of common ancestry leads to the suggestion that in the stock from which these inbred lines were ultimately derived, there were some 11.0 percent paired and 5.3 percent unpaired polymorphisms in the average mouse. This is about half the frequency of polymorphisms observed in wild European Mus musculus musculus and Mus musculus domesticus with one-dimensional electrophoresis of blood plasma and erythrocyte proteins. Three explanations were considered for the lower estimated frequency for liver protein polymorphisms: the difference is real, the apparent difference is due to the lower resolving power of two-dimensional gels, or the mouse strains from which the present inbred lines were drawn had already, lost through inbreeding, a considerable amount of their genetic variation before the inbreeding leading to the present strains commenced.     

Potent benzimidazolone based human beta(3)-adrenergic receptor agonists

The synthesis and biological evaluation of a series of benzimidazolone beta(3) adrenergic receptor agonists are described. A trend toward the reduction of rat atrial tachycardia upon increasing steric bulk at the 3-position of the benzimidazolone moiety was observed.     

Interaction of thrombin with sucrose octasulfate

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.