Control of protein levels is vital to cellular homeostasis, for maintaining a steady state, to coordinate changes during differentiation and other roles. In African trypanosomes surface proteins contribute to immune evasion, drug sensitivity and environmental sensing. The trypanosome surface is dominated by the GPI-anchored variant surface glycoprotein, but additional GPI-anchored and trans-membrane domain proteins are present with known roles as nutrient receptors and signal transducers. The evolutionarily conserved deubiquitinase orthologs of Usp7 and Vdu1 in trypanosomes modulate abundance of many surface proteins, including the invariant surface glycoproteins, which have roles in immune evasion and drug sensitivity. Here we identify multiple trypanosome Skp1 paralogs and specifically a divergent paralog SkpZ. Affinity isolation and LCMSMS indicates that SkpZ forms a heterotrimeric complex with TbUsp7 and TbTpr86, a tetratricopeptide-repeat protein. Silencing SkpZ decreases TbUsp7 and TbTpr86 abundance, confirming a direct association. Further, SkpZ knockdown decreases the abundance of multiple trans-membrane domain (TMD) proteins but increases GPI-anchored surface protein levels. Hence, a heterotrimeric complex of TbTpr86, TbUsp7 and SkpZ (TUSK) regulates expression levels of a significant cohort of trypanosome surface proteins mediating coordination between TMD and GPI-anchored protein expression levels.

Biology Centre Institute of Parasitology Czech Academy of Sciences České Budějovice Czechia

Department of Parasitology BIOCEV Charles University Vestec Czechia

School of Life Sciences University of Dundee Dundee United Kingdom

Zobrazit více v PubMed

Allen C. L., Liao D., Chung W. L., Field M. C. (2007). Dileucine signal-dependent and AP-1-independent targeting of a lysosomal glycoprotein in Trypanosoma brucei . Mol. Biochem. Parasitol. 156, 175–190. doi: 10.1016/j.molbiopara.2007.07.020 PubMed DOI

Allison H., O’Reilly A. J., Sternberg J., Field M. C. (2014). An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids. Microbial Cell 1, 325–345. doi: 10.15698/mic2014.10.170 PubMed DOI PMC

Alsford S., Horn D. (2008). Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei . Mol. Biochem. Parasitol. 161, 76–79. doi: 10.1016/j.molbiopara.2008.05.006 PubMed DOI PMC

Alsford S., Eckert S., Baker N., Glover L., Sanchez-Flores A., Leung K. F., et al. . (2012). High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature 482 (7384), 232–236. PubMed PMC

Baker N., Glover L., Munday J. C., Aguinaga Andrés D., Barrett M. P., de Koning H. P., et al. . (2012). Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. PNAS 109, 10996–11001. doi: 10.1073/pnas.1202885109 PubMed DOI PMC

Blatch G. L., Lässle M. (1999). The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. BioEssays 21, 932–939. doi: 10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO;2-N PubMed DOI

Chung W. L., Leung K. F., Carrington M., Field M. C. (2008). Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes. Traffic 9 (10), 1681–1697. PubMed

Das A. K., Cohen P. W., Barford D. (1998). The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for tpr-mediated protein-protein interactions. EMBO J. 2, 1192–1199. doi: 10.1093/emboj/17.5.1192 PubMed DOI PMC

Dean S., Sunter J., Wheeler R. J., Hodkinson I., Gluenz E., Gull K. (2015). A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 5, 140197. doi: 10.1098/rsob.140197 PubMed DOI PMC

Emmer B. T., Nakayasu E. S., Souther C., Choi H., Sobreira T. J., Epting C. L., et al. . (2011). Global analysis of protein palmitoylation in African trypanosomes. Eukaryotic Cell 10, 455–463. doi: 10.1128/EC.00248-10 PubMed DOI PMC

Gadelha C., Rothery S., Morphew M., McIntosh J. R., Severs N. J., Gull K. (2009). Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes. PNAS 106, 17425–17430. doi: 10.1073/pnas.0909289106 PubMed DOI PMC

Gadelha C., Zhang W., Chamberlain J. W., Chait B. T., Wickstead B., Field M. C. (2015). Architecture of a host-parasite interface: Complex targeting mechanisms revealed through proteomics. Mol. Cell. Proteomics 14, 1911–1926. doi: 10.1074/mcp.M114.047647 PubMed DOI PMC

Goldenberg S. J., Cascio T. C., Shumway S. D., Garbutt K. C., Liu J., Xiong Y., et al. . (2004). Structure of the Cand1-Cul1-Roc1 complex reveals regulatory mechanisms for the assembly of the multisubunit cullin-dependent ubiquitin ligases. Cell 12, 517–528. doi: 10.1016/j.cell.2004.10.019 PubMed DOI

Graf F. E., Ludin P., Wenzler T., Kaiser M., Brun R., Pyana P. P., et al. . (2013). Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol. PloS Negl. Trop. Dis. 10, e2475. doi: 10.1371/journal.pntd.0002475 PubMed DOI PMC

Gualdrón-López M., Chevalier N., van der Smissen P., Courtoy P. J., Rigden D. J., Michels P. A. M. (2013). Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features. Biochim. Biophys. Acta 1833, 3 076–33092. PubMed

Hao Y. H., Fountain M. D., Jr., Fon Tacer K., Xia F., Bi W., Kang S. H., et al. . (2015). USP7 acts as a molecular rheostat to promote WASH-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder. Mol. Cell. 59, 956–969. doi: 10.1016/j.molcel.2015.07.033 PubMed DOI PMC

Hershko A., Ciechanover A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 425–479. doi: 10.1146/annurev.biochem.67.1.425 PubMed DOI

Kessler B. M., Fortunati E., Melis M., Pals C. E., Clevers H., Maurice M. M. (2007). Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7. J. Proteome Res. 6, 4163–4172. doi: 10.1021/pr0702161 PubMed DOI

Kim R. Q., Sixma T. K. (2017). Regulation of USP7: A high incidence of E3 complexes. J. Mol. Biol. 10, 3395–3408. doi: 10.1016/j.jmb.2017.05.028 PubMed DOI

Lukeš J., Kachale A., Votýpka J., Butenko A., Field M. C. (2022). African trypanosome strategies for conquering new hosts and territories: the end of monophyly? Trends Parasitol. 38 (9), 724–736. PubMed

Macleod O. J. S., Cook A. D., Webb H., Crow M., Burns R., Redpath M., et al. . (2022). Invariant surface glycoprotein 65 of trypanosoma brucei is a complement C3 receptor. Nat. Commun. 13 (1), 5085. PubMed PMC

Makarov A., Began J., Mautone I. C., Pinto E., Ferguson L., Zoltner M., et al. . (2023). The role of invariant surface glycoprotein 75 in xenobiotic acquisition by african trypanosomes. Microb. Cell. 10 (2), 18–35. PubMed PMC

Melo do Nascimento L., Terrao M., Marucha K. K., Liu B., Egler F., Clayton C. (2020). The RNA-associated proteins MKT1 and MKT1L form alternative PBP1-containing complexes in trypanosoma brucei. J. Biol. Chem. 295 (32), 10940–10955. PubMed PMC

Mussmann R., Engstler M., Gerrits H., Kieft R., Toaldo C. B., Onderwater J., et al. . (2004). Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei . J. Biol. Chem. 24, 40690–40698. doi: 10.1074/jbc.M404697200 PubMed DOI

Nagai M., Shibata A., Ushimaru T. (2018). Cdh1 degradation is mediated by APC/C-Cdh1 and SCF-Cdc4 in budding yeast. Redox Biol. 18, 200–210. doi: 10.1016/j.bbrc.2018.10.179 PubMed DOI

Obado S. O., Field M. C., Chait B. T., Rout M. P. (2016). High-efficiency isolation of nuclear envelope protein complexes from trypanosomes. Methods Mol. Biol. 1411, 67–80. doi: 10.1007/978-1-4939-3530-7_3 PubMed DOI

Oberholzer M., Morand S., Kunz S., Seebeck T. (2006). A vector series for rapid PCR-mediated c-terminal in situ tagging of trypanosoma brucei genes. Mol. Biochem. Parasitol. 145 (1), 117–120. doi: 10.1016/j.molbiopara.2005.09.002 PubMed DOI

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D. J., et al. (2019). The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 8, D442–D450. doi: 10.1093/nar/gky1106 PubMed DOI PMC

Petroski M. D., Deshaies R. J. (2005). Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9–20. doi: 10.1038/nrm1547 PubMed DOI

Pettersen E. F., Goddard T. D., Huang C. C., Meng E. C., Couch G. S., Croll T. I., et al. . (2021). UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82. doi: 10.1002/pro.3943 PubMed DOI PMC

Rojas F., Koszela J., Búa J., Llorente B., Burchmore R., Auer M., et al. . (2017). The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei . PloS Negl. Trop. Dis. 13, e0005626. doi: 10.1371/journal.pntd.0005626 PubMed DOI PMC

Shimogawa M. M., Saada E. A., Vashisht A. A., Barshop W. D., Wohlschlegel J. A., Hill K. L. (2018). Cell surface proteomics provides insight into stage-specific remodeling of the host-parasite interface in Trypanosoma brucei . Mol. Cell Proteomics 14, 1977–1988. doi: 10.1074/mcp.M114.045146 PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M. Y., Geiger T., et al. . (2016). The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740. doi: 10.1038/nmeth.3901 PubMed DOI

Urbaniak M. D., Guther L. S., Ferguson M. A. J. (2012). Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages. PloS One 7, e36619. doi: 10.1371/journal.pone.0036619 PubMed DOI PMC

Venkatesh D., Zhang N., Zoltner M., Del Pino R. C., Field M. C. (2018). Evolution of protein trafficking in kinetoplastid parasites: Complexity and pathogenesis. Traffic 19 (11), 803–812. PubMed

Zheng N., Schulman B. A., Song L., Miller J. J., Jeffrey P. D., Wang P., et al. . (2002). Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 251, 703–709. doi: 10.1038/416703a PubMed DOI

Zoltner M., Leung K. F., Alsford S., Horn D., Field M. C. (2015). Modulation of the surface proteome through multiple ubiquitylation pathways in African trypanosomes. PloS Pathog. 22, e1005236. doi: 10.1371/journal.ppat.1005236 PubMed DOI PMC

Zoltner M., Del Pino R. C., Field M. C. (2020). Sorting the muck from the brass: Analysis of protein complexes and cell lysates. Methods Mol. Biol. 2116, 645–653. PubMed