The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

Mechanisms of G2 Arrest in Response to Overexpression of p53

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 and cyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions −287 to −123 of the cyclin B1 promoter and −104 to −74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway

PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden’s disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden’s disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4,5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.

A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N′-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

Fetal and maternal contributions to risk of pre-eclampsia: population based study

Objective: To use familial patterns of recurrence of pre-eclampsia to investigate whether paternal genes expressed in the fetus contribute to the mother’s risk of pre-eclampsia and whether mother’s susceptibility to pre-eclampsia is related to maternal inheritance by mitochondrial DNA.

trEST, trGEN and Hits: access to databases of predicted protein sequences

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits.isb-sib.ch).