Substrate search using recombinant activated LmxMPK4

The activation of LmxMPK4 by co-expression with LmxMKK5 does not only facilitate the development of inhibitor screenings against LmxMPK4, but provides also the possibility to search for the natural substrate of LmxMPK4 in L. mexicana. As previous studies have shown LmxMPK4 to be important in promastigote and amastigote stages (Wang, Q. et al.

2005) it can be presumed that the in vivo substrate of LmxMPK4 is constitutively present in Leishmania. The addition of activated recombinant LmxMPK4 and radioactively labelled ATP to Leishmania lysates should therefore lead to the phosphorylation of the sought substrate and consequently to a detectable band in an autoradiograph corresponding to the protein band of the in vivo substrate of LmxMPK4. To ensure the phosphotransferase reaction would use radioactively labelled ATP, all endogenous ATP was removed from the lysates. One amastigote lysate was additionally treated with λ-phosphatase to remove any already present phosphorylations on the proteins, as not to interfere with substrate phosphorylation by LmxMPK4. The quality of the obtained autoradiographs was surprisingly low with hardly any separate protein bands visible in the blurred lane, despite the fact that the lysates displayed perfectly separated proteins without much degradation in the Coomassie-stained gel (Fig. 18, right panels). The blurred picture appeared regardless of the addition of recombinant co-expressed LmxMPK4, His-LmxMPK4K59M or no protein to the lysates. Nevertheless, a distinct band at about 27 kDa was detected in all three examined lysate samples after the addition of activated LmxMPK4, but not in the presence of the kinase-dead mutant LmxMPK4K59M or without addition of recombinant protein. It can therefore be concluded that the phosphorylation of the protein which constitutes the band at 27 kDa is solely due to the activity of LmxMPK4 and not any bacterial or Leishmania kinase that was present in the lysates. MS analysis of the excised protein band ascertained the masses of several contained peptides. Matching the masses with a L. major protein database led to the identification of several proteins as

potential natural substrates of LmxMPK4. Only peptides which also corresponded in mass to peptides in the respective L. mexicana homologues were considered relevant. It is likely that the excised protein band was made up of several distinct proteins, as parasite lysates contain a large number of proteins which will obviously overlap in size and as the analysed gel piece was deliberately chosen to be rather too broad than to risk missing out on putative substrates. None of the peptides identified by MS analysis contained any phosphorylations on serine, threonine or proline, which makes it impossible to definitely say which one, if any, of the detected proteins are the in vivo substrate of LmxMPK4.

They do, however, provide us with a suggestion of possible interaction partners of LmxMPK4 in L. mexicana. Not one of the allocated proteins was detected in all three samples of promastigote, amastigote and dephosphorylated amastigote lysates. Proteins that were annotated by peptides found in only one analysed sample include the two hypothetical proteins LmjF36.2480 and LmjF34.3830 and tryparedoxin peroxidase (LmjF15.1120) all of which contain at least one potential MAP kinase phosphorylation site [S/T]-P (sequences including potential phosphorylation sites and annotated peptides shown in appendix). A regulatory MAP kinase phosphorylation site is expected to be conserved between L. major and L. mexicana, which is not the case for the SP-motif in tryparedoxin peroxidase that LmxM15.1160 misses. This makes it highly unlikely that tryparedoxin peroxidase is an in vivo substrate of LmxMPK4. The hypothetical protein LmxM36.2480 is equally out of the question, as the annotated L. major peptide does not correspond in mass to the homologous peptide sequence in L. mexicana. No statements can be made about the plausibility of the hypothetical protein LmjF34.3830/LmxM33.3830 being a LmxMPK4 substrate, apart from the fact that both proteins contain conserved SP-motifs. Two proteins were detected in two different samples, respectively. The γ-subunit of the ATP synthase F1 was identified in promastigote lysate samples as well as in the sample containing the dephosphorylated lysate of axenic amastigotes. The F1 enzyme is the cytoplasmic part of the F1F0-complex, which is responsible for the generation of energy by oxidative phosphorylation in most trypanosomatid life stages, apart from bloodstream trypanosomes where it hydrolyses ATP to generate the membrane potential (Schnaufer, A. et al. 2005). The ATP synthesis is driven by the proton electrochemical potential gradient and phosphorylations of subunits have so far not been linked with regulation of the enzyme-complex, which means that the γ-subunit of the ATP synthase F1

is an unlikely in vivo substrate for LmxMPK4. Moreover, does the sequence of the γ-subunit not include any SP or TP motifs, the typical substrate phosphorylation sites of MAP kinases and was therefore not further considered as a potential substrate of LmxMPK4. The only other protein, which was identified in more than one sample, was the glycosomal malate dehydrogenase (MDH) (LmjF19.0710/LmxM19.0710) with a size of 33.63 kDa, annotated in the promastigote lysate and the non-phosphatase treated amastigote sample. Four peptides in the untreated amastigote sample were identified as

malate dehydrogenase (LmjF34.0140), two of which also matched the L. mexicana homologue LmxM33.0140. LmjF34.0140 and LmxM33.0140 are not annotated with a subcellular localisation in gene.db, but are predicted to be localised in the mitochondrion by the program TargetP1.1 (Emanuelsson, O. et al. 2000). An alignment of these two, as well as the mitochondrial MDH is enclosed in the appendix and contains markings of all annotated peptides. The two peptides matching the L. major mitochondrial MDH did not match the L. mexicana homologue LmxM33.0160, which is why this protein was dismissed as potential substrate. None of the identified MDHs contain the ideal MAP kinase substrate phosphorylation sequence P-X-[S/T]-P, but they do each contain at least one motif with the reduced consensus sequence [S/T]-P (Clark-Lewis, I. et al. 1991;

Davis, R. J. 1993) that is conserved between L. major and L. mexicana. Unfortunately, none of the peptides identified by MS analysis contain any of the [S/T]-P motifs, so that there is no evidence on the phosphorylation status of these proteins in the Leishmania lysates after incubation with active LmxMPK4. Malate dehydrogenases play an important role in Leishmania metabolism, converting malate to oxaloacetate. Leishmania encodes MDHs with cytosolic, mitochondrial and glycosomal localisation. The mitochondrial MDH is part of the TCA cycle whose enzymes are mainly used for non-cyclic pathways in trypanosomatids. Mitochondrial MDH is therefore one of the enzymes involved in the generation of precursors for fatty acid biosynthesis and in the degradation of amino acids.

Malate is also transported through membranes and is a precursor for the generation of phosphoenolpyruvate (PEP), the starting point of gluconeogenesis. It has so far not been reported that MDH activity is regulated by phosphorylation, but as many metabolic enzymes are regulated by kinases and the protein contains potential MAP kinase phosphorylation sites, MDH is potentially a substrate for LmxMPK4 in vivo. The fact that LmxMPK4 and its T. brucei homologue TbMAPK2 are essential in Leishmania promastigotes and amastigotes, as well as in procyclic T. brucei forms suggests that the kinase plays a central role in the parasites and the regulation of MDH would be consistent with that. Moreover, as TbMAPK2 is not essential in bloodstream forms of T. brucei it can be concluded that either TbMAPK2 and LmxMPK4 play different roles in the parasites or that their function is essential in promastigotes, amastigotes and procyclics, but not in bloodstream forms. As bloodstream form trypanosomes rely solely on glycolysis for their energy metabolism they only express cytosolic malate dehydrogenase (Aranda, A. et al.

2006), which could be a possible explanation for the relative unimportance of TbMAPK2 in bloodstream trypanosomes, if mitochondrial and glycosomal MDH were the natural substrates of the kinase. However, considering the poor quality of samples used for MS analysis, the potential for miss-matches due to the usage of a L. major protein database and the fact that no phosphorylated peptides were detected, MDH as a potential in vivo substrate for LmxMPK4 remains purely speculative. Further experimental approaches are required before a definite conclusion can be drawn.

5.1.2 Characterisation of an inhibitor-sensitised mutant of