Proteomic consequences of a single gene mutation in a colorectal cancer model

The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation by using an isogenic CRC cell culture model in which wildtype APC expression has been ectopically restored. Using LC-MS/MS label free shotgun proteomics, over 5000 proteins were identified in SW480Null (mutant APC) and SW480APC (APC restored). We observed 155 significantly differentially expressed proteins between the two cell lines, with 26 proteins showing opposite expression trends relative to gene expression measurements. Protein changes corresponded to previously characterized features of the APCNull phenotype: loss of cell adhesion proteins, increase in cell cycle regulators, alteration in Wnt signaling related proteins, and redistribution of β-catenin. Increased expression of RNA processing and isoprenoid biosynthetic proteins occurred in SW480Null cells. Therefore, shotgun proteomics reveals proteomic differences associated with a single gene change, including many novel differences that fall outside known target pathways.     

A-signalling and the cell density requirement for Myxococcus xanthus development.

Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.     

A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus

The Myxococcus xanthus asg genes ( asgA, asgB , and asgC ) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG , and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD , the gene encoding the major sigma factor of M. xanthus . The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A-signal restores developmental expression of an A-signal-dependent gene (Ω4521) in the asgC767 ( rpoDEK598 ) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

Genes required for developmental signalling in Myxococcus xanthus: three asg loci.

asg-carrying strains of Myxococcus xanthus arose in a selection for mutants defective in cell-cell signalling during fruiting body development. All 15 asg mutations examined were found to lie in one of three genetic loci, asgA, asgB, or asgC. The loci were defined by linkage to different insertions of transposon Tn5 and molecular cloning of asgA. asg mutants of all three types were deficient in the aggregation of cells into mounds of the sort that normally give rise to fruiting bodies. asg mutants were also deficient in spore formation; sporulation is normally one of the last steps in fruiting body development. Consistent with a requirement for cell-to-cell signalling, at 1 to 2 h asg+-carrying cells release a material called A-factor that can rescue development of asg mutants. asgA, asgB, and asgC mutants released 5% or less of the asg+ level of A-factor, as measured by bioassay. The experimental results are consistent with the hypothesis that a deficiency in A-factor production or release is the primary developmental defect in asg mutants and that aggregation and sporulation depend on A-factor. asg mutations at all three loci also changed the color and morphology of growing colonies, and failure to release A-factor may itself arise from a defect in growing cells.     

Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus.

Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal. During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases. A DNA probe of the omega 4521 gene was constructed. Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal. A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation. To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated. All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521. Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg). sasA and sasB mutants differ phenotypically during growth and development. Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels. sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation. In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally. The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521.     

Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.     

An active variant of the prokaryotic transposable element IS903 carries an amber stop codon in the middle of an open reading frame

Summary A nematode mutant lacking pheromone activity does not enter the developmentally arrested dispersal stage called the dauer larva unless exogenous pheromone is added to the growth medium, indicating that the pheromone is required for wild-type dauer larva formation. In contrast, a class of temperature-sensitive mutant forms dauer larvae even in the absence of detectable pheromone, indicating that such mutants bypass the normal pheromone requirement. A rapid bioassay of pheromone produced by individual nematodes has been developed for genetic analysis of pheromone production.     

DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants.

In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated. The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication. It requires active RecA protein. We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process). In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication. Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I. The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity. Thus, the Rip bypass pathway requires active DNA polymerase I. Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA. An accompanying paper (Y. Cao and T. Kogoma, J. Bacteriol. 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.     

Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis

Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system.     

Isolation and characterization of proline peptidase mutants of Salmonella typhimurium

The proline requirement of Salmonella typhimurium strain proB25 can be satisfied by either of the peptides Leu-Pro or Gly-Pro-Ala. A mutant derivative of strain proB25 isolated by penicillin selection in medium containing Leu-Pro as proline source fails to use either Leu-Pro or Gly-Pro-Ala as a source of proline. This strain is a double mutant that lacks two aminoacyl-proline-specific peptidases. One of these enzymes (peptidase Q) catalyzes the rapid hydrolysis of Leu-Pro but does not hydrolyze Gly-Pro-Ala or poly-l-proline. Mutations at a site (pepQ) near metE lead to loss of this activity. The other peptidase (peptidase P) catalyzes the hydrolysis of Gly-Pro-Ala and poly-l-proline but is only weakly active with Leu-Pro as substrate. This enzyme is similar to aminopeptidase P previously described in Escherichia coli (16). Mutations at a locus (pepP) near serA lead to loss of this enzyme.     

Retarded growth of an Escherichia coli mutant deficient in spermidine synthase can be unspecifically repaired by addition of various polyamines

The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.     

Phosphorylation of the rat vesicular acetylcholine transporter

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.     

Mutational analysis of the hydrolytic activity of yeast RNA polymerase III.

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309-325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056-9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli beta-subunit sequences between Rif(r) clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T(5) terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3'-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.     

Bacteriophage T4 bypass31 mutations that make gene 31 nonessential for bacteriophage T4 replication: mapping bypass31 mutations by UV rescue experiments.

The product of gene 31 is normally required for assembly of the T4 capsid. Two mutations that each bypass that requirement are shown to be located at separate sites in gene 23, which encodes the major structural protein of the capsid. A second phenotypic effect that characterizes both bypass31 mutant strains is the ability to multiply in host-defective strains, such as hdB3-1 and groEL mutants, on which wild-type T4 is unable to assemble capsids. The genetic data indicate that both phenotypic effects are due to the bypass31 mutation. Elimination of the requirement for both the phage protein, gp31, and the host protein, GroEL, by either of two single mutations in gene 23 indicates that GroEL and gp31 are normally needed to interact with gp23 in capsid assembly of wild-type T4.     

Mutations in pimE restore lipoarabinomannan synthesis and growth in a Mycobacterium smegmatis lpqW mutant

Lipoarabinomannans (LAMs) and phosphatidylinositol mannosides (PIMs) are abundant glycolipids in the cell walls of all corynebacteria and mycobacteria, including the devastating human pathogen Mycobacterium tuberculosis. We have recently shown that M. smegmatis mutants of the lipoprotein-encoding lpqW gene have a profound defect in LAM biosynthesis. When these mutants are cultured in complex medium, spontaneous bypass mutants consistently evolve in which LAM biosynthesis is restored at the expense of polar PIM synthesis. Here we show that restoration of LAM biosynthesis in the lpqW mutant results from secondary mutations in the pimE gene. PimE is a mannosyltransferase involved in converting AcPIM4, a proposed branch point intermediate in the PIM and LAM biosynthetic pathways, to more polar PIMs. Mutations in pimE arose due to insertion of the mobile genetic element ISMsm1 and independent point mutations that were clustered in predicted extracytoplasmic loops of this polytopic membrane protein. Our findings provide the first strong evidence that LpqW is required to channel intermediates such as AcPIM4 into LAM synthesis and that loss of PimE function results in the accumulation of AcPIM4, bypassing the need for LpqW. These data highlight new mechanisms regulating the biosynthetic pathways of these essential cell wall components.