Ubiquitomics: An Overview and Future

Biomolecules, Год журнала: 2020, Номер 10(10), С. 1453 - 1453

Опубликована: Окт. 17, 2020

Covalent attachment of ubiquitin, a small globular polypeptide, to protein substrates is key post-translational modification that determines the fate, function, and turnover most cellular proteins. Ubiquitin exists as mono- or polyubiquitin chains involving multiple ways how ubiquitin C-termini are connected lysine, perhaps other amino acid side chains, N-termini proteins, often including branching chains. Understanding this enormous complexity in ubiquitination, so-called ‘ubiquitin code’, combination with ∼1000 enzymes involved controlling recognition, conjugation, deconjugation, calls for novel developments analytical techniques. Here, we review different headways field mainly driven by mass spectrometry chemical biology, referred “ubiquitomics”, aiming understand system’s biological diversity.

Язык: Английский

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Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival

Nature Communications, Год журнала: 2021, Номер 12(1)

Опубликована: Фев. 23, 2021

Abstract Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and transmission hosts, requiring stringent spatial temporal regulation. Here, we apply kinome-wide gene deletion tagging in Leishmania mexicana promastigotes to define protein kinases with transition roles. Whilst 162 are dispensable, 44 kinase genes refractory likely core required parasite replication. Phenotyping pooled mutants using bar-seq projection pursuit clustering reveal functional phenotypic groups involved differentiation from metacyclic promastigote amastigote, growth survival macrophages mice, colonisation sand fly motility. This unbiased interrogation function allows targeted investigation organelle-associated signalling pathways successful parasitism.

Язык: Английский

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The pathogenicity and virulence of Leishmania - interplay of virulence factors with host defenses

Virulence, Год журнала: 2022, Номер 13(1), С. 903 - 935

Опубликована: Май 9, 2022

Leishmaniasis is a group of disease caused by the intracellular protozoan parasite genus Leishmania. Infection different species Leishmania results in various host immune responses, which usually lead to clearance and may also contribute pathogenesis and, hence, increasing complexity disease. Interestingly, tends reside within unfriendly environment macrophages has evolved survival strategies evade or modulate defense. This can be attributed array virulence factors vicious parasite, target important functioning machineries. review encompasses holistic overview leishmanial factors, their role assisting parasite-mediated evasion defense weaponries, modulating epigenetic landscapes regulatory genes. Furthermore, discusses diagnostic potential advent immunomodulators as futuristic antileishmanial drug therapy.

Язык: Английский

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Targeted Protein Degradation for Infectious Diseases: from Basic Biology to Drug Discovery

ACS Bio & Med Chem Au, Год журнала: 2022, Номер 3(1), С. 32 - 45

Опубликована: Дек. 15, 2022

Targeted protein degradation (TPD) is emerging as one of the most innovative strategies to tackle infectious diseases. Particularly, proteolysis-targeting chimera (PROTAC)-mediated may offer several benefits over classical anti-infective small-molecule drugs. Because their peculiar and catalytic mechanism action, PROTACs might be advantageous in terms efficacy, toxicity, selectivity. Importantly, also overcome emergence antimicrobial resistance. Furthermore, have potential (i) modulate "undruggable" targets, (ii) "recycle" inhibitors from drug discovery approaches, (iii) open new scenarios for combination therapies. Here, we try address these points by discussing selected case studies antiviral first-in-class antibacterial PROTACs. Finally, discuss how field PROTAC-mediated TPD exploited parasitic Since no antiparasitic PROTAC has been reported yet, describe parasite proteasome system. While its infancy with many challenges ahead, hope that diseases lead development next-generation

Язык: Английский

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Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets

Frontiers in Cellular and Infection Microbiology, Год журнала: 2022, Номер 12

Опубликована: Янв. 24, 2022

Leishmaniasis is one of the major public health concerns in Latin America, Africa, Asia, and Europe. The absence vaccines for human use lack effective vector control programs make chemotherapy main strategy to all forms disease. However, high toxicity available drugs, limited choice therapeutic agents, occurrence drug-resistant parasite strains are challenges related chemotherapy. Currently, only a small number drugs leishmaniasis treatment, including pentavalent antimonials (Sb V ), amphotericin B its formulations, miltefosine, paromomycin sulphate, pentamidine isethionate. In addition drug toxicity, failure serious concern. parasites causes closely diversity this genus. Owing enormous plasticity genome, resistance can occur by altering different metabolic pathways, demonstrating that mechanisms multifactorial extremely complex. Genetic variability genome cause not have limitations, but also search new challenging. Here, we examined biological characteristics hinder discovery.

Язык: Английский

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Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

PLoS Pathogens, Год журнала: 2020, Номер 16(10), С. e1008784 - e1008784

Опубликована: Окт. 27, 2020

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur Leishmania parasite differentiates between its main morphological forms, promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in mexicana genome but, currently, little is known about role of E1, enzymes this parasite. Bar-seq analysis 23 HECT/RBR null mutants generated promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes to amastigote differentiation mammalian infection. The E2s UBC1/CDC34, UBC2 UEV1 HECT HECT2 required successful transformation from UBA1b, UBC9, UBC14, HECT7 HECT11 normal proliferation during mouse Of all enzyme examined screen, Δ ubc2 uev1 exhibited most extreme differentiation. Null could not UBA1a or UBC3, UBC7, UBC12 UBC13, suggesting these essential promastigotes. X-ray crystal structure UEV1, orthologues human UBE2N UBE2V1/UBE2V2 respectively, reveal a heterodimer with highly conserved interface. Furthermore, recombinant L . load onto UBC2, allowing UBC2-UEV1 form K63-linked di-ubiquitin chains vitro Notably, cooperate E3s RNF8 BIRC2 non-K63-linked polyubiquitin chains, showing facilitate independent but association inhibits ability. Our study demonstrates dual essentiality intracellular survival shows interaction two proteins crucial regulation their activity function.

Язык: Английский

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Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase, and a cytosine base editor

Опубликована: Фев. 11, 2025

The ability to analyse the function of all genes in a genome is highly desirable, yet challenging Leishmania due repetitive genome, limited DNA repair mechanisms and lack RNA interference most species. While our introduction cytosine base editor (CBE) demonstrated potential overcome these limitations (Engstler Beneke (2023)), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, competition between deleterious non-deleterious mutations. Here, we present an optimized approach addressing issues.We identified T7 RNAP promoter variant ensuring high species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed prioritize STOP codon generation. Additionally, triple-expression construct created for stable integration sgRNA expression cassettes into safe harbor locus using AsCas12a ultra-mediated double-strand breaks, increasing efficiency by ∼400-fold one transfectant per 70 transfected cells. Using this improved small-scale proof-of-principle pooled screen, successfully confirmed essential fitness-associated functions CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140 RAB5A L. mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, hybrid CRISPR gene replacement within same cell line.Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Язык: Английский

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Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor

eLife, Год журнала: 2025, Номер 13

Опубликована: Фев. 24, 2025

The ability to analyze the function of all genes in a genome is highly desirable, yet challenging Leishmania due repetitive genome, limited DNA repair mechanisms, and lack RNA interference most species. While our introduction cytosine base editor (CBE) demonstrated potential overcome these limitations (Engstler Beneke, 2023), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, competition between deleterious non-deleterious mutations. Here, we present an optimized approach addressing issues. We identified T7 RNAP promoter variant ensuring high species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed prioritize STOP codon generation. Additionally, triple-expression construct created for stable integration sgRNA expression cassettes into safe harbor locus using AsCas12a ultra-mediated double-strand breaks, increasing efficiency by ~400-fold 1 transfectant per 70 transfected cells. Using this improved small-scale proof-of-principle pooled screen, successfully confirmed essential fitness-associated functions CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140, RAB5A mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, RNAP, hybrid CRISPR gene replacement within same cell line. Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Язык: Английский

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Structure and Activity of the Essential UCH Family Deubiquitinase DUB16 from Leishmania donovani

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 1, 2025

Abstract In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential parasite survival in promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from donovani , which belongs to C-terminal hydrolase (UCH) family. efficiently hydrolyses aminocoumarin rhodamine conjugates consistent with proposed cellular roles UCH-type DUBs regenerating free monomeric small molecule adducts arising adventitious metabolic processes. The crystal structure reveals typical deubiquitinase fold, relatively short disordered crossover loop appears restrict access catalytic cysteine. At close stoichiometric enzyme substrate ratios, exhibits activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues proximal C-terminus distal ubiquitin. With 100-1000-fold higher turnover rates, cleaves ubiquitin-ribosomal L40 fusion protein give mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits activity. IMP-1710 shown viability assays killing EC 50 values 1−2 μM, comparable anti-leishmanial drug, miltefosine. L. mexicana parasites engineered overproduce showed modest increase resistance providing evidence vivo . Together these results suggest on-target may be druggable target develop new anti-leishmania compounds.

Язык: Английский

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Update on relevant trypanosome peptidases: Validated targets and future challenges

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Год журнала: 2020, Номер 1869(2), С. 140577 - 140577

Опубликована: Ноя. 30, 2020

Язык: Английский

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