Thamir M. Ismail

Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells.

The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (NpnN) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The normal level of 3 μm adenylated NpnN (ApnN) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular NpnN in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

Background: Certain S100 proteins induce cell migration and metastasis but the molecular mechanism is not clear. Results: S100P preferentially binds and disperses NMIIA fibers and subsequently affects focal adhesion sites (FAS) and cell adhesion. Conclusion: S100P overexpression reduces the assembly of NMIIA-FAS to enhance cell migration by reducing anchoring forces. Significance: The mechanism of the primary step of S100P-induced metastasis has been elucidated. S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, Kd = 0.5 μm; IIB, Kd = 8 μm; IIC, Kd = 1.0 μm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

The basic C-terminal amino acids of calcium-binding protein S100A4 promote metastasis

The calcium-binding protein S100A4 can induce a metastatic phenotype in animal model systems and its expression in various human cancers has been shown to be associated with metastasis and reduced patient survival. Using a series of nested deletion mutants, it is now shown that the two C-terminal lysine residues are required for the enhanced metastasis, invasion and migration abilities that S100A4 confers on cells in a model system of metastasis. Basic C-terminal residues enhance the affinity between S100A4 and its best characterized target, a recombinant C-terminal fragment of non-muscle myosin II heavy chain isoform A (NMMHC-IIA). In wild-type S100A4 protein, the presence of the C-terminal lysine, residue 101, enhances the rate of association between S100A4 and NMMHC-IIA. These results identify the amino acids of S100A4 that are involved in metastasis induction and show that the C-terminal region of S100A4 is a possible target for inhibitors of its metastatic action.

Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation.

Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short‐flanking homology (SFH) or long‐flanking homology (LFH) gene replacement. Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs. Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S‐methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur‐containing amino‐acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response. The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10‐fold under sulphate starvation. Replacement cassettes, comprising the kanMX marker flanked by each ORFs promoter and terminator regions, were cloned into pUG7. All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap‐repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright

Functional analysis of six novel ORFs on the left arm of Chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short‐flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. Complementation of the yll035w deletion with its cognate clone, and a promoter‐substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonstrated that YLL035w is under cell‐cycle regulation. Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non‐essential ORFs. Replacement cassettes, comprising the kanMX marker flanked by each ORF’s promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap‐repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright

Functional analysis of eight open reading frames on chromosomes XII and XIV of Saccharomyces cerevisiae

Deletion, together with basic functional and bioinformatic analyses has been carried out on eight novel ORFs discovered during the sequencing of the Saccharomyces cerevisiae genome. Six ORFs (YLL049w, YLL051c, YLL052c, YLL053c, YLL054c and YLL055w) located on the left arm, and one (YLR130c) on the right arm, of chromosome XII, and an eighth ORF (YNL331c) on the left arm of the chromosome XIV, have been investigated. ORFs were deleted by the SFH–PCR gene‐replacement strategy. Basic functional analysis revealed no obvious phenotype for any of the eight ORFs. Bioinformatic analysis, however, revealed possible functions for seven of the ORFs on the basis of the amino acid sequence similarity of their predicted protein products to those of proteins with known functions. ORF YLL051c (FRE6) shows similarity to iron transport proteins, such as ferric reductase. YLL052c and YLL053c appear to be aquaporins. The product of YLL054c (Yll054p) is highly similar to the oleate‐specific transcriptional activator protein (Pip2p), which is involved in the peroxisomal induction pathway (pip). ORF YLL055w is similar to Dal5p, allantoate permease, and may play role in allantoin transport. YLR130c (ZRT2) is a low‐affinity zinc transporter protein. YNL331c is also named AAD14, which is induced by chemicals that induce oxidative stress by depleting the cell of glutathione. Copyright

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae

Deletion via PCR‐mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading‐frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short‐flanking homology (SFH) or a long‐flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non‐growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non‐essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap‐repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright

S100A4 elevation empowers expression of metastasis effector molecules in human breast cancer

Many human glandular cancers metastasize along nerve tracts, but the mechanisms involved are generally poorly understood. The calcium-binding protein S100A4 is expressed at elevated levels in human cancers, where it has been linked to increased invasion and metastasis. Here we report genetic studies in a Drosophila model to define S100A4 effector functions that mediate metastatic dissemination of mutant Ras-induced tumors in the developing nervous system. In flies overexpressing mutant RasVal12 and S100A4, there was a significant increase in activation of the stress kinase JNK and production of the matrix metalloproteinase MMP1. Genetic or chemical blockades of JNK and MMP1 suppressed metastatic dissemination associated with S100A4 elevation, defining required signaling pathway(s) for S100A4 in this setting. In clinical specimens of human breast cancer, elevated levels of the mammalian paralogs MMP2, MMP9, and MMP13 are associated with a 4- to 9-fold relative decrease in patient survival. In individual tumors, levels of MMP2 and MMP13 correlated more closely with levels of S100A4, whereas MMP9 levels correlated more closely with the S100 family member S100P. Overall, our results suggest the existence of evolutionarily conserved pathways used by S100A4 to promote metastatic dissemination, with potential prognostic and therapeutic implications for metastasis by cancers that preferentially exploit nerve tract migration routes. Cancer Res; 77(3); 780-9. ©2016 AACR.

Activation of tissue plasminogen activator by metastasis-inducing S100P protein

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 μM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity.