New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.
The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.
Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay.
Mariana
M
Margenat
Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay.
Anne-Marie
AM
Labandera
Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, CP 11400, Uruguay.
Magdalena
M
Gil
Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, CP 11400, Montevideo, Uruguay.
Federico
F
Carrion
Departamento de Bioquímica, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, Brazil.
Marcela
M
Purificação
Departamento de Bioquímica, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, Brazil.
Guilherme
G
Razzera
1] Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay [2] Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, CP 11400, Uruguay [3] Instituto de Investigaciones Biológicas Clemente Estable, CP 11600, Montevideo, Uruguay.
María Magdalena
MM
Portela
Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, CP 11400, Montevideo, Uruguay.
Gonzalo
G
Obal
Departamento de Bioquímica, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, Brazil.
Hernán
H
Terenzi
Unidad de Biofísica de Proteínas, Institut Pasteur de Montevideo, CP 11400, Montevideo, Uruguay.
Otto
O
Pritsch
Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, CP 11400, Uruguay.
Rosario
R
Durán
Cátedra de Inmunología, Facultad de Ciencias-Facultad de Química, Universidad de la República, CP 11600, Montevideo, Uruguay.
Ana María
AM
Ferreira
Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay.
Andrea
A
Villarino
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology*; D013058:Mass Spectrometry; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; C414465:MptpA protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases