Securin is not required for chromosomal stability in human cells

Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin^−/− cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.     

The integrity of tubulin molecule is not required for the activity of tubulin carboxypeptidase

Tubulin dimer, alpha-tubulin subunit, and C-terminal peptides obtained from the alpha-tubulin subunit were compared in their capabilities to act as substrates of tubulin carboxypeptidase. The results obtained indicate that the enzyme does not require the beta-tubulin subunit to release tyrosine from alpha-tubulin. The 17-Kd C-terminal peptide of the alpha-tubulin subunit was obtained and it was detyrosinated at the same rate as tubulin dimer. A smaller C-terminal peptide of 2.8-3.7 Kd showed a lower capability to act as substrate. Similar results were obtained with pancreatic carboxypeptidase A. From the analysis of the results we consider that an optimal activity of the tubulin carboxypeptidase depends mainly on the accessibility of the C-terminal end of alpha-tubulin.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

Primary structure of aspergillopepsin I deduced from nucleotide sequence of the gene and aspartic acid-76 is an essential active site of the enzyme for trypsinogen activation

The coding region of the aspergillopepsin I (EC 3.4.23.18) gene occupies 1340 base pairs of the genomic DNA and is separated into four exons by three intros. The predicted amino-acid sequence of aspergillopepsin I consists of 325 residues and is 32% and 27% homologous with those of human pepsin and calf chymosin. The cDNA of the gene prepared from mRNA has been cloned and expressed in yeast cells. To identify the residue of the substrate binding pocket in determining the specificity of aspergillopepsin I towards basic substrates, this residue was replaced with a serine residue by site-directed mutagenesis. The mutation is a single amino-acid change, Asp-76 converted to Ser-D76S, in the enzyme. The striking feature of this is that only the trypsinogen activating activity was destroyed. We therefore concluded that Asp-76 is the binding site towards basic substrates.     

Mitochondrial ferredoxin is required for heme A synthesis in Saccharomyces cerevisiae.

Heme A is a prosthetic group of all eukaryotic and some prokaryotic cytochrome oxidases. This heme differs from heme B (protoheme) at two carbon positions of the porphyrin ring. The synthesis of heme A begins with farnesylation of the vinyl group at carbon C-2 of heme B. The heme O product of this reaction is then converted to heme A by a further oxidation of a methyl to a formyl group on C-8. In a previous study (Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) FEBS Lett. 492, 133-138) we proposed that the formyl group is formed by an initial hydroxylation of the C-8 methyl by a three-component monooxygenase consisting of Cox15p, ferredoxin, and ferredoxin reductase. In the present study three lines of evidence confirm a requirement of ferredoxin in heme A synthesis. 1) Temperature-conditional yah1 mutants grown under restrictive conditions display a decrease in heme A relative to heme B. 2) The incorporation of radioactive delta-aminolevulinic acid into heme A is reduced in yah1 ts but not in the wild type after the shift to the restrictive temperature; and 3) the overexpression of Cox15p in cytochrome oxidase mutants that accumulate heme O leads to an increased mitochondrial concentration of heme A. The increase in heme A is greater in mutants that overexpress Cox15p and ferredoxin. These results are consistent with a requirement of ferredoxin and indirectly of ferredoxin reductase in hydroxylation of heme O.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.     

Intracellular aspartic proteinase Apr1p of Candida albicans is required for morphological transition under nitrogen-limited conditions but not for macrophage killing

Vacuolar hydrolases have been thoroughly characterized in Saccharomyces cerevisiae, but their homologues in the fungal pathogen Candida albicans have received less attention. The genes APR1 and CPY1 of C. albicans encode putative vacuolar aspartic proteinase and serine carboxypeptidase, respectively. We examined properties of apr1Δ and cpy1Δ mutants, showing that Cpy1p molecular species detected in cell lysates of apr1Δ and its parental strain did not differ in molar mass. Processing of Cpy1p precursor is apparently independent of Apr1p. This is in contrast to S. cerevisiae, where vacuolar aspartic proteinase Pep4p is known to participate in the activation of other vacuolar hydrolases including serine carboxypeptidase. We also found that both apr1Δ and cpy1Δ strains are able to form hyphae in nutrient-rich filamentation media. However, proline as a sole nitrogen source induced filamentation only in cpy1Δ and its parental strain, but not in apr1Δ. This indicates the importance of Apr1p for the morphological transition under nitrogen-limited conditions. Despite that, the ability of apr1Δ to kill murine macrophages was not reduced under the conditions tested.     

Securin is required for chromosomal stability in human cells

Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.     

Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.

The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.     

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.     

Conserved cysteine 126 in triosephosphate isomerase is required not for enzymatic activity but for proper folding and stability

In triosephosphate isomerase, Cys126 is a conserved residue located close to the catalytic glutamate, Glu165. Although it has been mentioned that Cys126 and other nearby residues are required to maintain the active site geometry optimal for catalysis, no evidence supporting this idea has been reported to date. In this work, we studied the catalytic and stability properties of mutants C126A and C126S of Saccharomyces cerevisiae TIM (wtTIM). None of these amino acid replacements induced significant changes in the folding of wtTIM, as indicated by spectroscopic studies. C126S and C126A have K(M) and k(cat) values that are concomitantly reduced by only 4-fold and 1.5-fold, respectively, compared to those of wtTIM; in either case, however, the catalytic efficiency (k(cat)/K(M)) of the enzyme is barely affected. The affinity of mutated TIMs for the competitive inhibitor 2-phosphoglycolate augmented also slightly. In contrast, greater susceptibility to thermal denaturation resulted from mutation of Cys126, especially when it was changed to Ser. By using values of the rate constants for unfolding and refolding, we estimated that, at 25 degrees C, C126A and C126S are less stable than wtTIM by about 5.0 and 9.0 kcal mol(-)(1), respectively. Moreover, either of these mutations slows down the folding rate by a factor of 10 and decreases the recovery of the active enzyme after thermal unfolding. Thus, Cys126 is required for proper stability and efficient folding of TIM rather than for enzymatic catalysis.     

Drosophila ankyrin 2 is required for synaptic stability

Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, we recovered mutations in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability.     

Exogenous fibronectin is not required for organogenesis in vitro

Summary The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50μg/ml) was a strong mitogen for kidney cells, whereas, the addition of soluble fibronectin (50 to 250 μg/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. however, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 μg/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250μg/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro. This work was supported by grants from the Sigrid Jusélius Foundation, Finska L?kares?llskapet, Finnish Life Insurance Companies and Grant CA 28896 and Cancer Center Support Grant CA 30199 from the National Cancer Institute, Department of Health and Human Services, Washington, D.C.     

Exogenous fibronectin is not required for organogenesis in vitro

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.     

Apoptosis is not required for acantholysis in pemphigus vulgaris

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory proteins FLIP(L) and FLIP(S) to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.     

The kinesin-associated protein UNC-76 is required for axonal transport in the Drosophila nervous system

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.