The VSFASSQQ motif confers calcium sensitivity to the intracellular apyrase LALP70

Biederbick, Annette; Rösser, Ralf; Storre, Jörg; Elsässer, Hans–Peter

doi:10.1186/1471-2091-5-8

Cited by 11 publications

(10 citation statements)

References 28 publications

(27 reference statements)

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“…MDC has been described as a specific autophagic vacuole marker (Biederbick et al, 1995), but since this compound also acts as a lysosomotropic agent, MDC could eventually label some acidic compartments derived form the endocytic pathway. Therefore, we used monodansylpentane (MDH) that is a new derivative of MDC (Niemann et al, 2001).…”

Section: Autophagic Vacuoles Are Decorated By Egfp-rab7mentioning

confidence: 99%

“…For this purpose we generated stably transfected CHO cells that overexpress the chimeric proteins EGFP-Rab7 wt and the mutants Q67L and T22N. Autophagic vacuoles were labeled with the autofluorescent drug monodansylcadaverine (MDC), a specific autophagosomal marker (Biederbick et al, 1995;Munafo and Colombo, 2001). By fluorescence microscopy we observed that, in living cells transfected with pEGFP-Rab7 wt and the active mutant Rab7Q67L (Fig.…”

Section: Autophagic Vacuoles Are Decorated By Egfp-rab7mentioning

confidence: 99%

“…Autophagic vacuoles were labeled with monodansylcadaverine (MDC) by incubating cells grown on coverslips with 0.05 mM MDC in PBS at 37°C for 10 minutes (Biederbick et al, 1995;Munafo and Colombo, 2001). In some experiments cells were labeled with a MDC derivative, monodansylpentane (MDH; 0.1 mM in PBS) (Niemann et al, 2001).…”

Section: Visualization Of Mdc-or Mdh-labeled Vacuolesmentioning

confidence: 99%

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Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Gutierrez

Munafó

Berón

et al. 2004

Journal of Cell Science

592

493

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Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.

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Section: Autophagic Vacuoles Are Decorated By Egfp-rab7mentioning

confidence: 99%

Section: Autophagic Vacuoles Are Decorated By Egfp-rab7mentioning

confidence: 99%

Section: Visualization Of Mdc-or Mdh-labeled Vacuolesmentioning

confidence: 99%

See 1 more Smart Citation

Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Gutierrez

Munafó

Berón

et al. 2004

Journal of Cell Science

592

493

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“…7). To determine whether these vacuoles were autophagic vacuoles, infected cells were labelled with an autofluorescent drug, MDC, a selective marker for autophagic vacuoles (Biederbick et al, 1995). MDC-labelled vesicles were not evident in HeLa cells infected with wild-type (Fig.…”

Section: Parahaemolyticus Does Not Induce Autophagy In Hela Cellsmentioning

confidence: 99%

Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines

2009

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The Vibrio parahaemolyticus type III secretion system 1 (T3SS1) induces cytotoxicity in mammalian epithelial cells. We characterized the cell death phenotype in both epithelial (HeLa) and monocytic (U937) cell lines following infection with V. parahaemolyticus. Using a combination of the wild-type strain and gene knockouts, we confirmed that V. parahaemolyticus strain NY-4 was able to induce cell death in both cell lines via a T3SS1-dependent mechanism. Bacterial contact, but not internalization, was required for T3SS1-induced cytotoxicity. The mechanism of cell death involves formation of a pore structure on the surface of infected HeLa and U937 cells, as demonstrated by cellular swelling, uptake of cell membrane-impermeable dye and protection of cytotoxicity by osmoprotectant (PEG3350). Western blot analysis showed that poly ADP ribose polymerase (PARP) was not cleaved and remained in its full-length active form. This result was evident for seven different V. parahaemolyticus strains. V. parahaemolyticus-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) or the caspase-1 inhibitor N-acetyl-tyrosyl-valylalanyl-aspartyl-aldehyde (Ac-YVAD-CHO); thus, caspases were not involved in T3SS1-induced cytotoxicity. DNA fragmentation was not evident following infection and autophagic vacuoles were not observed after monodansylcadaverine staining. We conclude that T3SS1 of V. parahaemolyticus strain NY-4 induces a host cell death primarily via oncosis rather than apoptosis, pyroptosis or autophagy. INTRODUCTIONVibrio parahaemolyticus is a Gram-negative food-borne pathogen that is commonly associated with consumption of raw or undercooked seafood (Joseph et al., 1982). Symptoms of infection include diarrhoea, nausea, vomiting, headache, fever and chills. In addition to typical gastrointestinal infections, approximately 5 % of V. parahaemolyticus infections advance to septicaemia (Hlady & Klontz, 1996) and these infections may be fatal, especially in immunocompromised patients or those with a preexisting medical condition such as liver disease or diabetes (Yeung & Boor, 2004).Thermostable direct haemolysin (TDH) is considered the primary virulence factor in V. parahaemolyticus. In addition to its ability to form pores in red blood cells, TDH causes increased short circuit current, increased Cl 2 secretion in human colonic epithelial cells, and enhanced Ca 2+ entry from the extracellular medium (Takahashi et al., 2001). An early study of a tdh deletion mutant demonstrated significantly reduced ability to cause fluid accumulation in ileal loops of a rabbit model (Nishibuchi et al., 1992). In contrast, Park et al. (2004) showed that a tdh deletion mutant has the ability to cause fluid accumulation in the ligated intestine of rabbits. Furthermore, both TDH-positive and -negative strains of V. parahaemolyticus can disrupt the epithelial tight junction, a precursor to dissemination of bacteria into the circulation (Lynch et a...

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“…The LALP70 mRNA contains an additional stretch of 24 bp, which encodes 8 amino acids inserted in-frame into the UDPase protein sequence (30)(31)(32). This additional 8-residue motif was reported to confer calcium sensitivity to LALP70 (33), and the apyrase products of the two splice variants were shown to possess somewhat different enzymatic properties (30). The two NTPDASE4-encoded proteins differ also in their reported cellular localization.…”

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confidence: 99%

NTPDASE4 Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death

Avital-Shacham

Sharf

Kleinberger

2014

J Virol

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The adenovirus E4orf4 protein induces nonclassical apoptosis in mammalian cells through at least two complementing pathways regulated by the interactions of E4orf4 with protein phosphatase 2A (PP2A) and Src kinases. In Saccharomyces cerevisiae cells, which do not express Src, E4orf4 induces PP2A-dependent toxicity. The yeast Golgi apyrase Ynd1 was found to contribute to E4orf4-mediated toxicity and to interact with the PP2A-B55␣ regulatory subunit. In addition, a mammalian Ynd1 orthologue, the NTPDASE4 gene product Golgi UDPase, was shown to physically interact with E4orf4. Here we report that knockdown of NTPDASE4 suppressed E4orf4-induced cell death. Conversely, overexpression of the NTPDASE4 gene products Golgi UDPase and LALP70 enhanced E4orf4-induced cell killing. We found that similarly to results obtained in yeast, the apyrase activity of mammalian UDPase was not required for its contribution to E4orf4-induced toxicity. The interaction between E4orf4 and UDPase had two consequences: a PP2A-dependent one, resulting in increased UDPase levels, and a PP2A-independent outcome that led to dissociation of large UDPase-containing protein complexes. The present report extends our findings in yeast to E4orf4-mediated death of mammalian cells, and combined with previous results, it suggests that the E4orf4-NTPDase4 pathway, partly in association with PP2A, may provide an alternative mechanism for the E4orf4-Src pathway to contribute to the cytoplasmic death function of E4orf4. IMPORTANCEThe adenovirus E4orf4 protein contributes to regulation of the progression of virus infection from the early to the late phase, and when expressed alone, it induces a unique caspase-independent programmed cell death which is more efficient in cancer cells than in normal cells. The interactions of E4orf4 with cellular proteins that mediate its functions, such as PP2A and Src kinases, are highly conserved in evolution. The results presented here reveal that the NTPDASE4 gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in Saccharomyces cerevisiae, associates with E4orf4 and plays a role in induction of cell death in mammalian cells. Details of the functional interaction between E4orf4, PP2A, and the UDPase are described. Identification of the evolutionarily conserved mechanisms underlying E4orf4 activity will increase our understanding of the interactions between the virus and the host cell and will contribute to our grasp of the unique mode of E4orf4-induced cell death.

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The VSFASSQQ motif confers calcium sensitivity to the intracellular apyrase LALP70

Cited by 11 publications

References 28 publications

Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines

NTPDASE4 Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death

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