Neil Q. McDonald

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinsons disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinsons disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinsons disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinsons disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.

S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu catalytic triad [Murray-Rust, J., Leiper, J. M., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P. & McDonald, N. (2001) Nat. Struct. Biol. 8, 679–683]. The presence of a reactive cysteine residue (Cys-249) in the active site of DDAH raised the possibility that DDAH activity might be directly regulated by S-nitrosylation of this residue by NO. In the present study, we demonstrate that recombinant DDAH is reversibly inhibited after incubation with NO donors in vitro. Similarly mammalian DDAH in cytosolic extracts is also reversibly inhibited by NO donors. In cultured endothelial cells, heterologously expressed human DDAH II was S-nitrosylated after cytokine induced expression of the inducible NOS isoforms. The implication of these findings is that under certain conditions when NO generation increases, S-nitrosylation diminishes DDAH activity and this would be expected to lead to accumulation of asymmetric dimethylarginine and inhibition of NOS. This observation may help explain why expression of iNOS often leads to inhibition of activity of constitutively expressed NOS isozymes. We also identify Cys-His-Glu as a nitrosylation motif that is conserved in a family of arginine handling enzymes.

Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase

Nitric oxide synthase is inhibited by asymmetric NG-methylated derivatives of arginine whose cellular levels are controlled in part by dimethylarginine dimethylaminohydrolase (DDAH, EC 3.5.3.18). Levels of asymmetric NG,NG-dimethylarginine (ADMA) are known to correlate with certain disease states. Here, the first structure of a DDAH shows an unexpected similarity to arginine:glycine amidinotransferase (EC 2.1.4.1) and arginine deiminase (EC 3.5.3.6), thus defining a superfamily of arginine-modifying enzymes. The identification of a Cys-His-Glu catalytic triad and the structures of a Cys to Ser point mutant bound to both substrate and product suggest a reaction mechanism. Comparison of the ADMA–DDAH and arginine–amidinotransferase complexes reveals a dramatic rotation of the substrate that effectively maintains the orientation of the scissile bond of the substrate with respect to the catalytic residues. The DDAH structure will form a basis for the rational design of selective inhibitors, which are of potential use in modulating NO synthase activity in pathological settings.

Structure and chemical inhibition of the RET tyrosine kinase domain.

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors.

A heterozygous effect for PINK1 mutations in Parkinson's disease?

To investigate the significance of PINK1 mutations in sporadic Parkinsons disease (PD).

Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation.

The crystal structure of the catalytic domain from the MAPK phosphatase Pyst1 (Pyst1–CD) has been determined at 2.35 Å. The structure adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active site that displays a distorted geometry in the absence of its substrate with some similarity to the dual–specificity phosphatase cdc25. Functional characterization of Pyst1–CD indicates it is sufficient to dephosphorylate activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1–CD using the substrate p–nitrophenyl phosphate (pNPP) reveals that both molecules undergo catalytic activation in the presence of recombinant inactive ERK2, switching from a low– to high–activity form. Mutation of Asp 262, located 5.5 Å distal to the active site, demonstrates it is essential for catalysis in the high–activity ERK2–dependent conformation of Pyst1 but not for the low–activity ERK2–independent form, suggesting that ERK2 induces closure of the Asp 262 loop over the active site, thereby enhancing Pyst1 catalytic efficiency.

SAC1 Encodes a Regulated Lipid Phosphoinositide Phosphatase, Defects in Which Can Be Suppressed by the Homologous Inp52p and Inp53p Phosphatases

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1mutants. Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins. We have now demonstrated this activity for Sac1p and have characterized its specificity. The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations. Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant. However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest. It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.

The Dual Mechanism of Separase Regulation by Securin

BACKGROUND Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown. RESULTS We show that, in budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the proteins C terminus. CONCLUSIONS Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securins own destruction at anaphase onset.

Crystal structure of the C2 domain from protein kinase C-δ

BACKGROUND The protein kinase C (PKC) family of lipid-dependent serine/theonine kinases plays a central role in many intracellular eukaryotic signalling events. Members of the novel (delta, epsilon, eta, theta) subclass of PKC isotypes lack the Ca2+ dependence of the conventional PKC isotypes and have an N-terminal C2 domain, originally defined as V0 (variable domain zero). Biochemical data suggest that this domain serves to translocate novel PKC family members to the plasma membrane and may influence binding of PKC activators. RESULTS The crystal structure of PKC-delta C2 domain indicates an unusual variant of the C2 fold. Structural elements unique to this C2 domain include a helix and a protruding beta hairpin which may contribute basic sequences to a membrane-interaction site. The invariant C2 motif, Pro-X-Trp, where X is any amino acid, forms a short crossover loop, departing radically from its conformation in other C2 structures, and contains a tyrosine phosphorylation site unique to PKC-delta. This loop and two others adopt quite different conformations from the equivalent Ca(2+)-binding loops of phospholipase C-delta and synaptotagmin I, and lack sequences necessary for Ca2+ coordination. CONCLUSIONS The N-terminal sequence of Ca(2+)-independent novel PKCs defines a divergent example of a C2 structure similar to that of phospholipase C-delta. The Ca(2+)-independent regulation of novel PKCs is explained by major structural and sequence differences resulting in three non-functional Ca(2+)-binding loops. The observed structural variation and position of a tyrosine-phosphorylation site suggest the existence of distinct subclasses of C2-like domains which may have evolved distinct functional roles and mechanisms to interact with lipid membranes.