Structural And Functional Studies Of Ubiquitin-like Protein TbUfm1 From Trypanosoma Brucei

As an important class of post-translational modifications,ubiquitin-like protein(Ubl)modification is involved in numerous biological processes and regulates complex cellular events.Ubls share low sequence identity,but their structures are highly conserved.Similar to ubiquitin modification pathway,Ubls are covalently conjugated to target proteins through a three-step enzymatic reaction,thereby affecting the function of the substrate proteins.As one of the Ubls,ubiquitin-fold modifier 1(Ufml)is highly conserved in eukaryotes except for yeast.So far,the main identified function of Ufml and Ufmylation system is that Ufm1 system can be induced by endoplasmic reticulum(ER)stress and participates in the ER stress response.In addition,Ufml and Ufmylation also plays important roles in embryonic development,erythroid progenitor differentiation,tumor growth,and so on,but the mechanisms of these functions are still unclear.Trypanosoma brucei(T.brucei)is the pathogen of African trypanosomiasis,and is also the model organism for eukaryotic biological research,however,little is known about the Ufml and Ufmylation in T.brucei.In order to provide some new directions for research of Ufm1,we investigated the structure and function of Ufml in T.brucei in this study.Sequence alignment showed that there exists a homolog of Ufml in T.brucei,which is named as TbUfm1.Through affinity purification of cell line expressing TbUfml and mass spectrometry analysis,about 100 TbUfml-conjugated and-associated proteins were identified.Among these proteins,homologs of E1,E2,and E3 of Ufm1(named as TbUba5,TbUfc1 and TbUfll,respectively)were identified.Homolog of Ufml-specific cleavage enzyme 2(TbUfsp2)was also identified by sequence alignment.These four proteins were proved to be the E1,E2,E3,and Ufsp2 of TbUfml system in T.brucei respectively by enzymatic reactions in vitro,and the catalytic cysteine at residue 271 of TbUba5 and residue 115 of TbUfcl were necessary for the enzymatic reaction in vitro.Meanwhile,we determined the solution structure of TbUfm1.Structure of TbUfml is composed of three ?-helixes and four ?-sheets,and its central helix al and all ?-sheets constitute the typical Ubl fold.Compared to HsUfm1,TbUfm1 has a longer N-terminal extension,a shorter ?3 sheet,and an extra short ?2 helix.NMR chemical shift perturbation of TbUfm1 titrated with TbUba5,TbUfc1,TbUfl1 showed that TbUfml binds to TbUba5 in vitro through a hydrophobic surface composed of its ?1?2?1?2.HsUfml also binds to HsUba5,and the most binding sites of TbUfm1 are similar to those of HsUfm1.Furthermore,investigation of the function of Ufml and Ufmylation in T.brucei was conducted.Among the TbUfml-conjugated and-associated proteins,about one quarter are the cytoskeleton proteins.The cytoskeleton of cell lines expressing TbUfm1,TbUba5,TbUfc1,and TbUfl1 were extracted.The results showed TbUfm1,TbUba5,TbUfcl and TbUfll all have partial cytoskeletal distribution,which implies that TbUfml and its system may be involved in cytoskeleton-related cell events.Among TbUfml-conjugated and-associated proteins,there is a conserved substrate named TbUfbpl(homolog of HsUfbpl)and a trypanosomatid-specific substrate named TbCAP80.TbUfbpl can be modified by TbUfm1 in vitro.HsUfbpl is crucial in maintaining the endoplasmic reticulum homeostasis,suggesting that TbUfml and Ufmylation may be involved in the same cellular event.TbCAP80 is a multifunctional protein,which is necessary for cell growth.Depletion of TbUfml and TbCAP80 impaired the fluid-phase endocytosis to uptake dextran,indicating a conserved and essential role of Ufml in vesicle trafficking.TbUfml and TbCAP80 are both localized in glycosome in T.brucei,which implies that TbUfml may be related to glycolysis.To prove it,cell energy metabolites before and after the depletion of TbUfml were detected.The results showed that the depletion of TbUfml decreased the production of NADH and fumarate.Besides,in the starvation stress,TbUfm1-modified proteins were up-regulated quickly.These results demonstrated the roles of Ufml in glycolysis and starvation stress response.In human 293 cells,HsUfml is co-localized with HsALD(Fructose-biosphosphate aldolase,the marker protein of glycosome)in starvation stress,implying that human Ufml might be also involved in energy metabolism and starvation stress response.This function might be conserved for Ufm1 from trypanosoma to human.Overall,our work not only revealed the conserved role of TbUfm1 and Ufmylation in regulating vesicle trafficking,but also shed light on Ufml's novel roles in regulating glycolysis,energy metabolism and stress response over starvation stress.