An unusual casein kinase 1 from Trypanosoma cruzi epimastigotes

Abstract

Background: CK1 enzymes are serine/threonine protein kinases that regulate numerous cellular processes. Although seven putative CK1 ortholog genes have been identified in the genome of Trypanosoma cruzi, the causative agent of Chagas' disease, only two parasite CK1 isoforms have been characterized to date. Methods: T. cruzi epimastigotes were collected at the exponential phase of growth. The parasite clarified cytosolic fraction was separated by anion-exchange column chromatography, and the non-adsorbed fraction was rechromatographed on a second anion-exchange column. In order to identify the parasite casein kinases, ATP:phosphotransferase activity was evaluated by using dephosphorylated casein as substrate and [γ -32 P] ATP as cosubstrate. In addition, specific peptide substrates and inhibitors for higher eukaryotic CK1 and CK2 enzymes were employed to classify the purified T. cruzi protein kinase. Results: A soluble protein kinase that uses casein as a substrate, and possesses an apparent molecular weight of 33,000, was purified from the exponential phase of growth of T. cruzi epimastigotes. The purified protein specifically phosphorylated P1 (sequence=RRKDLHDDEEDEAMSITA), a selective peptide substrate for CK1, but not P2 (sequence=RRRADDSDDDDD) which is a CK2 substrate, and was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide and 1-(8-chloro-5-isoquinolinesulfonyl)piperazine, two specific inactivators of CK1. Consequently, the purified casein kinase was classified as a CK1 enzyme. Kinetic studies showed that the T. cruzi CK1 has a K m of 172.5±5.1 µM, 0.2062 ± 0.0051 mg/ml and 35.5±2.9 μM for ATP, casein and P1, respectively. Interestingly, this CK1 was stimulated in the presence of about 0.1 mM GTP or 5'-guanylylimidodiphosphate, but was inhibited at approximately 1 mM of either of these guanine nucleotides. Additionally, this enzyme was more than 80% inactivated by low concentrations of heparin (1 μM), which is a common inhibitor of CK2 enzymes. However, other inhibitors of mammalian CK2, such as emodin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, only affected the parasite CK1 activity at the highest concentrations used. Our findings demonstrate that this CK1 from T. cruzi has a dual modulation by GTP and an unusual sensitivity for heparin. Conclusions: A novel CK1 activity that functionally differs from previously characterized T. cruzi CK1 enzymes was purified from the exponential phase of growth of epimastigote forms.

Phylobioactive hotspots identified through multidimensional profiling of botanical drugs used to treat Chagas disease in Bolivia and Dioscorides’ De Materia Medica

“…In our study, emodin showed specific submicromolar inhibition of parasite release in CHO cells. Emodin was previously only tested on epimastigotes at high micromolar concentrations (94) where it inhibited casein kinase 1 with an IC 50 value of 130 µM (95), thus significantly less potent than in the trypomastigote infection assay shown here. Other reported effects of this natural product include antiinflammatory, antiosteoporosis, and antidepressant effects (93).…”

Phylobioactive hotspots in plant resources used to treat Chagas disease

“…In our study, emodin showed specific inhibition of T. cruzi parasite release in CHO cells in the nanomolar or low micromolar range, depending on host cell culture condition. Emodin has been tested previously only on epimastigotes at high micromolar concentrations ( De Lima et al, 2017 ) and as weak inhibitor of casein kinase 1 with an IC 50 value of 130 μM ( Justiniano et al, 2014 ). Other reported effects of emodin include anti-inflammatory, antiosteoporosis, anti-cardiovascular disease, and antidepressant effects ( Li and Jiang, 2018 ).…”

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates

“…For instance, CK substrate CK-S (sequence = (RRKHAAIG(pS)AYSITA, where pS corresponds to phosphorylated serine) is a substrate for CK2 in L. donovani , [102]. Pep1 (sequence = RRKDLHDDEEDEAMSITA) and Pep2 (sequence = RRRADDSDDDDD) are selective substrates for CK1 and CK2, respectively, and not only have been used as substrates for new kinase activity, but have also been used to sub-classify CKs identified from parasites kinetoplastids [104-109, 111–113] and apicomplexans [33, 110, 114]. DDDEESITRR and KRRRAL(pS)VASLPGL [110] have also been used as CK1-specific peptide substrates, and RRREEE TEEE [110], RRREDEESDDEE, eIF2β-derived peptide MSGDEMIFDPTMSKKKKKKKKP [114] and RRASADDSDDEDL [115] have also been used as a CK2-specific peptide substrates.…”

“…DDDEESITRR and KRRRAL(pS)VASLPGL [110] have also been used as CK1-specific peptide substrates, and RRREEE TEEE [110], RRREDEESDDEE, eIF2β-derived peptide MSGDEMIFDPTMSKKKKKKKKP [114] and RRASADDSDDEDL [115] have also been used as a CK2-specific peptide substrates. Typical Pep1 concentrations for activity assays are 100 – 800 μM [104-108, 113], while Pep2 concentrations usually range 40 -200 μM [33, 104–109, 112, 113]. Recombinant CK1.1 from T. cruzi had apparent Km for β-casein of 5.7 mg/mL and for Pep1 of 128 μM [108].…”