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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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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Experimental structure based drug design (SBDD) applications for anti‐leishmanial drugs: A paradigm shift?

Medicinal Research Reviews, Год журнала: 2023, Номер 44(3), С. 1055 - 1120

Опубликована: Дек. 24, 2023

Leishmaniasis is a group of neglected tropical diseases caused by at least 20 species Leishmania protozoa, which are spread the bite infected sandflies. There three main forms disease: cutaneous leishmaniasis (CL, most common), visceral (VL, also known as kala-azar, serious), and mucocutaneous leishmaniasis. One billion people live in areas endemic to leishmaniasis, with an annual estimation 30,000 new cases VL more than 1 million CL. New treatments for urgent need, existing ones inefficient, toxic, and/or expensive. We have revised experimental structure-based drug design (SBDD) efforts applied discovery drugs against grouped explored targets according metabolic pathways they belong to, key achieved advances highlighted evaluated. In cases, SBDD studies follow high-throughput screening campaigns secondary pharmacokinetic optimization, due majoritarian belief that there few validated However, some strategies significantly contributed candidates bigger number holds promise future development.

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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Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania Mexicana

Molecular and Biochemical Parasitology, Год журнала: 2024, Номер 258, С. 111619 - 111619

Опубликована: Март 29, 2024

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin ubiquitin-like modifiers to target proteins. The extent of the proteasome system in Leishmania mexicana its importance for parasite survival has recently been established through deletion mutagenesis life-cycle phenotyping studies. conjugating E2 UBC2, variant UEV1, with it forms a stable complex vitro, were shown be essential differentiation promastigote parasites infectious amastigote form. To investigate further, we used immunoprecipitation Myc-UBC2 or Myc-UEV1 identify interacting proteins L. promastigotes. interactome UBC2 comprises multiple ubiquitin-proteasome components including UEV1 four RING E3 ligases, as well potential substrates predicted have roles carbohydrate metabolism intracellular trafficking. smaller six proteins, shared consistent presence UBC2-UEV1 complexes. Recombinant RING1, RING2 RING4 ligases support transfer reactions involving E1, UBA1a, available substrate unanchored chains. These studies define additional UBC2-dependent ubiquitination pathway previously differentiation.

Язык: Английский

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Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase

Frontiers in Cellular and Infection Microbiology, Год журнала: 2021, Номер 11

Опубликована: Ноя. 10, 2021

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, development LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed more straightforward effective editing. this system, plasmid pTB007 is delivered episomal expression or integration β-tubulin locus stable T7 RNA polymerase Cas9. South America, especially Brazil, ( Viannia ) braziliensis most frequent etiological agent tegumentary leishmaniasis. The L. presents significant sequence divergence comparison major , which precludes To overcome limitation, sequences, present pTB007, were replaced by conserved pTB007_Viannia plasmid. This modification successful cassette M2903 genome, silico predictions suggest that can also be achieved other species. activity Cas9 was evaluated knocking out flagellar PF16, caused phenotype immobility transfectants. Endogenous PF16 successfully mNeonGreen, an in-locus complementation strategy employed return C-terminally copy original locus, resulted recovery swimming capacity. modified both provided important tool study biology parasite.

Язык: Английский

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Ubiquitin and ubiquitin-like conjugation systems in trypanosomatids

Current Opinion in Microbiology, Год журнала: 2022, Номер 70, С. 102202 - 102202

Опубликована: Сен. 11, 2022

In eukaryotic cells, reversible attachment of ubiquitin and ubiquitin-like modifiers (Ubls) to specific target proteins is conducted by multicomponent systems whose collective actions control protein fate cell behaviour in precise but complex ways. trypanosomatids, Ubls regulates the cycle, endocytosis, sorting degradation, autophagy various aspects infection stress responses. The extent these trypanosomatids has been surveyed recent reports, while Leishmania mexicana, essential roles have defined for many ubiquitin-system genes deletion mutagenesis life-cycle phenotyping campaigns. first steps elucidate pathways transfer among ubiquitination components define acceptor substrates downstream deubiquitinases are now being taken.

Язык: Английский

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Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin

Scientific Reports, Год журнала: 2021, Номер 11(1)

Опубликована: Ноя. 8, 2021

Abstract The inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene ( ubc13) of Plasmodium falciparum study its role in ubiquitylation and functional consequence during parasite asexual blood stage . Deletion resulted a significant reduction growth vitro , reduced Lys63 residue ubiquitin attached protein substrates, an increased sensitivity both mutagen, methyl methanesulfonate antimalarial drug dihydroartemisinin (DHA), but not chloroquine. also sensitive UBC13 inhibitor NSC697923. data suggest that this does code for conjugating responsible K63 ubiquitylation, which is important DNA repair pathways as previously demonstrated other organisms. DHA absence ubc13 function indicates may act primarily through pathway inhibitors enhance efficacy directly inhibit growth.

Язык: Английский

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