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Mechanism of atypical ubiquitination and deubiquitination by bacterial effectors

细菌效应子的非典型泛素化和去泛素化机制

基本信息

批准号：
10737296
负责人：
Chittaranjan Das
金额：
$ 46.83万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-16 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10737296
关键词：
ADP ribosylation Actins Address Amino Acids Arginine Attenuated Autophagocytosis Back Bacteria Binding Binding Sites Biochemical Biological Assay Calmodulin Cell physiology Complement Consumption Cryoelectron Microscopy Crystallography Databases Deubiquitination Economic Development Enzymes Eukaryota Excision Family Family member Generations Gram-Negative Bacteria Hand Hydrolase Hydrolysis Integration Host Factors Legionella Legionella pneumophila Legionnaires&apos Disease Life Cycle Stages Light Lysine Maintenance Molecular Mono(ADP-Ribose) Transferases Natural regeneration Nutrient Orthologous Gene Pathway interactions Phagosomes Poisoning Prevention strategy Process Property Proteins Public Health Reaction Regulation Resistance Serine Side Signal Transduction Sorting Structure Toxin Tubular formation Ubiquitin Ubiquitination Vesicle Virulence Work X-Ray Crystallography appendage cofactor enzyme activity hydroxyl group infectious disease treatment insight novel novel strategies paralogous gene particle pathogen pathogenic bacteria phosphoric diester hydrolase ribose phosphate structural determinants trafficking ubiquitin isopeptidase virtual

项目摘要

The pathogenic bacterium responsible for Legionnaires’ disease, Legionella pneumophila, uses SidE family of effectors (such as SdeA) to target several host proteins through a noncanonical ubiquitination mechanism radically different from the ATP-driven, E1-E2-E3 ubiquitination of eukaryotes. This mechanism involves an all- in-one ubiquitination machinery in SdeA which employs, first, mono-ADP-ribosylation (mART) of ubiquitin (Ub) at Arg42, catalyzed by its mART domain, to produce ADP-ribosylated ubiquitin (ADPR-Ub), which is then subjected an additional catalytic step, executed by the phosphodiesterase (PDE) activity embedded in a separate domain, resulting in phosphoribosyl (PR) ubiquitination of serine residues of host targets. Essential to the pathogen’s intracellular life cycle, SdeA and its orthologs target numerous host proteins involved in a range of processes, from vesicular trafficking to nutrient acquisition and autophagy. While resistant to host deubiquitinases, the PR ubiquitination is regulated at multiple levels at the hands of other effectors: the SidJ effector (and its paralog SdjA) can shut off mART activity by modifying a key catalytic residue though a pseudokinase-based polyglutamylation activity; whereas the DupA and DupB effectors can reverse PR- ubiquitination by restoring host targets (such as Rab33) to their native form. This sort of deubiquitination activity, while releasing the native host target, still leaves Ub as a modified derivative, with a phosphoribosyl appendage at Arg42 (PR-Ub). Accumulation of such a Ub derivative, that cannot be used in host ubiquitination pathways, has the effect of poisoning the cellular Ub pool which could be detrimental to Legionella’s replication. In this proposal we explore regeneration of free, functional Ub from PR-Ub through a two-step process involving an unusual AMPylation reaction catalyzed by a novel S-HxxxE motif-containing, actin-activated AMPylator, called LnaB, producing ADPR-Ub, which is then further processed by a macrodomain (ADP- ribosyl)hydrolase, MavL, returning Ub to its native form. Using single particle cryo-EM we will provide structural basis of actin activation, PR-Ub recognition and the ATP binding site of LnaB. The EM studies will be complemented with x-ray crystallography of apo LnaB and its ATP-bound form. Together with biochemical studies aimed at capturing enzyme intermediates, our work will provide key insights into the novel AMPylation reaction. The MavL effector, while using macrodomain for deADP-ribosylation, features a unique motif which we found was shared by a group of previously uncharacterized proteins in the DUF4804 family of the Pfam database. Such a motif appears to confer residue-level selectivity for arginine de-ADP ribosylation, a novel aspect of macrodomain function. We seek to provide structural basis of ADPR-Ub recognition, while elucidating the basis of arginine selectivity across the newly found MavL-like enzymes. Collectively, our study will reveal a novel mechanism for AMPylation, that appears to be employed by a large family of toxins of unknown function from diverse pathogens, while expanding the scope of macrodomain hydrolases.

军团病的致病细菌，嗜肺军团菌，使用SidE家族

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Genetically Encoded Crosslinking Enables Identification of Multivalent Ubiquitin-Deubiquitylating Enzyme Interactions.

基因编码交联能够鉴定多价泛素-去泛素化酶相互作用。

DOI：
10.1002/cbic.202300305
发表时间：
2023
期刊：
Chembiochem : a European journal of chemical biology
影响因子：
0
作者：
Patel,Rishi;NegrónTerón,Kristos;Zhou,Mowei;Nakayasu,Ernesto;Drown,Bryon;Das,Chittaranjan
通讯作者：
Das,Chittaranjan

The Two Deubiquitinating Enzymes from Chlamydia trachomatis Have Distinct Ubiquitin Recognition Properties.

DOI：
10.1021/acs.biochem.9b01107
发表时间：
2020-04-28
期刊：
Biochemistry
影响因子：
2.9
作者：
Hausman JM;Kenny S;Iyer S;Babar A;Qiu J;Fu J;Luo ZQ;Das C
通讯作者：
Das C

Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.

对细菌去泛素酶SDEA催化的水解中泛素产物释放的见解。

DOI：
10.1021/acs.biochem.0c00760
发表时间：
2021-03-02
期刊：
Biochemistry
影响因子：
2.9
作者：
Sheedlo MJ;Kenny S;Podkorytov IS;Brown K;Ma J;Iyer S;Hewitt CS;Arbough T;Mikhailovskii O;Flaherty DP;Wilson MA;Skrynnikov NR;Das C
通讯作者：
Das C

The unity of opposites: Strategic interplay between bacterial effectors to regulate cellular homeostasis.