The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Abstract

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.Trypanosoma brucei causes human African trypanosomiasis or African sleeping sickness, which is marked by debilitating neurologic symptoms ranging from sensory impairment to the characteristic aberrant sleeping patterns that progress to coma. If untreated, human African trypanosomiasis is fatal. With 30,000 deaths a year and 60 million people living at risk (1), human African trypanosomiasis is a major disease burden in sub-Saharan Africa. Current drugs are ineffective and toxic, and drug resistance is becoming a growing hurdle for treatment (2).T. brucei alternates between human and tsetse fly hosts, requiring extensive and rapid physiologic adaptations. In humans, the major T. brucei population consists of the extracellular, proliferative slender form in the bloodstream, which irreversibly differentiates into the G 1 -arrested stumpy form poised for transmission to the tsetse fly. Taken up by the tsetse fly, the stumpy form differentiates into the proliferative procyclic form in the insect midgut. Eventually, the tsetse salivary gland becomes populated with metacyclic forms, which infect the human host (3). This differentiation cycle requires survival in a diverse set of environments and forms the basis for infectivity and transmission.The molecular signals, regulators, and effectors underlying this complex sequence of events are not well understood but could provide novel targets for therapeutic interference. Distinct patterns of protein tyrosine phosphorylation accompany and often precede stage progression (4), suggesting that tyrosine phosphorylation is a key mechanism of developmental regulation. Studies of the T. brucei dual specificity kinases also provide evidence that tyrosine phosphorylation regulates the trypanosome life cycle (5-7). Moreover, NOPP44/46, 2 a nucleolar RNA-binding protein required for ribosome biogenesis (8), exhibits dramatic ch...

Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas’ disease

“…Analysis of the protein interfaces in the crystal lattice using PDBePISA[56] did not reveal any strong indications that the structures form a dimer in solution. Indeed, the elution profile from size exclusion chromatography demonstrated that TcPTP1 migrates as a monomer (data not shown), as was also observed for the classical PTPase from Tryapnosoma brucei , TbPTP1b[48]. Since the structures of chain A and chain B are very similar, the coordinates of chain A were used for structural analysis.…”

“…=1.0 Å over 274 aligned residues, 64% sequence identity, PDB code: 3m4u)[48] and human PTP1B (1.8 Å over 251 aligned atoms, 27% sequence identity, PDB code: 2hnq)[61]. Structural alignments of TcPTP1 and these two homologs reveal that the secondary structure elements align well with most of the structural deviations existing in the loops that connect them (Fig.…”

“…A monomer of the Trypanosoma brucei protein tyrosine phosphatase TbPTP1 (PDB code: 3m4u) was selected as a search model based on 64% sequence identity with TcPTP1 and prepared for molecular replacement by removing all solvent molecules and side chains of non-identical residues[48]. The search model was used to locate two molecules in the asymmetric unit with Phaser and data extending to 3.0 Å resolution.…”

The Potential of Secondary Metabolites from Plants as Drugs or Leads against Protozoan Neglected Diseases—Part III: In-Silico Molecular Docking Investigations

“…Plasmepsin II (PMII) 1SME [224]; 1LEE, 1LF2 [225]; 1LF3, 1LF4 [226]; 2BJU [227]; 2IGX, 2IGY [228]; 3F9Q [229]; 1M43 [230]; 1ME6 [231]; 1W6H, 1W6I [232]; 1XDH, 1XE5, 1XE6 [233] Plasmepsin IV (PMIV) 1LS5 [225] Proline racemase (PRACA) 1W61, 1W62 [234] Protein Kinase 5 (PK5) 1OB3, 1V0O, 1V0P [235] Protein tyrosine phosphatase 1 (PTP1) 3M4U [236] 4AZ1 [237] Pteridine reductase 1 (PTR1) 2XOX [238] 1E7W, 1E92 [239]; 1W0C [240]; 2BF7, 2BFA,2BFM, 2BFO, 2BFP [241]; 2QHX, 3H4V [242] 2C7V [243]; 2WD7, 2WD8, 3GN1, 3GN2 [244]; 2VZ0 [245]; 3BMC, 3BMN, 3BMO, 3BMQ, 3JQ6, 3JQ7, 3JQ8, 3JQ9, 3JQA, 3JQB, 3JQC, 3JQD, 3JQE, 3JQF, 3JQG [246]; 2X9N, 2X9G, 2X9V, 3MCV [247]; 2YHI [248] Pteridine reductase 2 (PTR2) 1MXF, 1MXH [249] Purine nucleoside phosphorylase (PNP) 1NW4, 1Q1G [250]; 2BSX, 1SQ6 [251]; 3ENZ [252] Pyridoxal kinase (PdxK) 3ZS7 [253] Pyruvate kinase (PYK)…”

Finding the Smoking Gun: Protein Tyrosine Phosphatases as Tools and Targets of Unicellular Microorganisms and Viruses

“…The crosstalk of TbPTP1 and TbPIP39 can be abolished by the differentiation regulators citrate and cis-aconitate [190]. The crystal structure of TbPTP1 is available [192]. The activity of TbPTP1 is sensitive to ROS, and can be completely inhibited in vitro by 10 mM sodium orthovanadate (90% inhibition using 1 mM sodium orthovanadate), but not ≤50 mM sodium fluoride.…”