title: Trypanosomatid RACK1 orthologs show functional differences associated with translation despite similar roles in Leishmania pathogenesis.
abstract: RACK1 proteins belong to the eukaryote WD40-repeat protein family and function as spatial regulators of multiple cellular events, including signaling pathways, the cell cycle and translation. For this latter role, structural and genetic studies indicate that RACK1 associates with the ribosome through two conserved positively charged amino acids in its first WD40 domain. Unlike RACK1s, including Trypanosoma brucei RACK1 (TbRACK1), only one of these two positively-charged residues is conserved in the first WD40 domain of the Leishmania major RACK1 ortholog, LACK. We compared virulence-attenuated LACK single copy (LACK/-) L. major, with L. major expressing either two LACK copies (LACK/LACK), or one copy each of LACK and TbRACK1 (LACK/TbRACK1), to evaluate the function of these structurally distinct RACK1 orthologs with respect to translation, viability at host temperatures and pathogenesis. Our results indicate that although the ribosome-binding residues are not fully conserved in LACK, both LACK and TbRACK1 co-sedimented with monosomes and polysomes in LACK/LACK and LACK/TbRACK1 L. major, respectively. LACK/LACK and LACK/TbRACK1 strains differed in their sensitivity to translation inhibitors implying that minor sequence differences between the RACK1 proteins can alter their functional properties. While biochemically distinguishable, both LACK/LACK and LACK/TbRACK1 lines were more tolerant of elevated temperatures, resistant to translation inhibitors, and displayed robust pathogenesis in vivo, contrasting to LACK/- parasites.
authors.affiliation: Department of Microbiology Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America.
authors.forename: Kohelia
authors.initials: K
authors.lastname: Choudhury
authors.forename: Daviel
authors.initials: D
authors.lastname: Cardenas
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Pullikuth
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Catling
authors.forename: Ashok
authors.initials: A
authors.lastname: Aiyar
authors.forename: Ben L
authors.initials: BL
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D005260:Female; D018320:Leishmania major / Q000166:cytology / Q000472:pathogenicity* / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D012269:Ribosomal Proteins; C093139:LACK antigen, Leishmania