Publications by authors named "Juliana B T Carnielli"

7 Publications

  • Page 1 of 1

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

mBio 2021 06 15;12(3):e0068721. Epub 2021 Jun 15.

York Biomedical Research Institute, Department of Biology, University of York, Heslington, United Kingdom.

During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.
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http://dx.doi.org/10.1128/mBio.00687-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262961PMC
June 2021

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors.

Nat Microbiol 2020 10 13;5(10):1207-1216. Epub 2020 Jul 13.

Novartis Institute for Tropical Diseases, Emeryville, CA, USA.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.
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http://dx.doi.org/10.1038/s41564-020-0745-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610364PMC
October 2020

Natural Resistance of to Miltefosine Contributes to the Low Efficacy in the Treatment of Visceral Leishmaniasis in Brazil.

Am J Trop Med Hyg 2019 10;101(4):789-794

Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil.

In India, visceral leishmaniasis (VL) caused by has been successfully treated with miltefosine with a cure rate of > 90%. To assess the efficacy and safety of oral miltefosine against Brazilian VL, which is caused by , a phase II, open-label, dose-escalation study of oral miltefosine was conducted in children (aged 2-12 years) and adolescent-adults (aged 13-60 years). Definitive cure was assessed at a 6-month follow-up visit. The cure rate was only 42% (6 of 14 patients) with a recommended treatment of 28 days and 68% (19 of 28 patients) with an extended treatment of 42 days. The in vitro miltefosine susceptibility profile of intracellular amastigote stages of the pretreatment isolates, from cured and relapsed patients, showed a positive correlation with the clinical outcome. The IC mean (SEM) of eventual cures was 5.1 (0.4) µM, whereas that of eventual failures was 12.8 (1.9) µM ( = 0.0002). An IC above 8.0 µM predicts failure with 82% sensitivity and 100% specificity. The finding of amastigotes resistant to miltefosine in isolates from patients who eventually failed treatment strongly suggests natural resistance to this drug, as miltefosine had never been used in Brazil before this trial was carried out.
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http://dx.doi.org/10.4269/ajtmh.18-0949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779219PMC
October 2019

A Leishmania infantum genetic marker associated with miltefosine treatment failure for visceral leishmaniasis.

EBioMedicine 2018 Oct 27;36:83-91. Epub 2018 Sep 27.

Centre for Immunology and Infection, Department of Biology, University of York, United Kingdom.; Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom.. Electronic address:

Background: Miltefosine has been used successfully to treat visceral leishmaniasis (VL) in India, but it was unsuccessful for VL in a clinical trial in Brazil.

Methods: To identify molecular markers that predict VL treatment failure whole genome sequencing of 26 L. infantum isolates, from cured and relapsed patients allowed a GWAS analysis of SNPs, gene and chromosome copy number variations.

Findings: A strong association was identified (p = 0·0005) between the presence of a genetically stable L. infantumMiltefosine Sensitivity Locus (MSL), and a positive response to miltefosine treatment. The risk of treatment failure increased 9·4-fold (95% CI 2·11-53·54) when an isolate did not have the MSL. The complete absence of the MSL predicted miltefosine failure with 0·92 (95% CI 0·65-0·996) sensitivity and 0·78 (95% CI 0·52-0·92) specificity. A genotyping survey of L. infantum (n = 157) showed that the frequency of MSL varies in a cline from 95% in North East Brazil to <5% in the South East. The MSL was found in the genomes of all L. infantum and L. donovani sequenced isolates from the Old World (n = 671), where miltefosine can have a cure rate higher than 93%.

Interpretation: Knowledge on the presence or absence of the MSL in L. infantum will allow stratification of patients prior to treatment, helping to establish better therapeutic strategies for VL treatment. FUND: CNPq, FAPES, GCRF MRC and Wellcome Trust.
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http://dx.doi.org/10.1016/j.ebiom.2018.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197651PMC
October 2018

Proteomic analysis of the soluble proteomes of miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates obtained from Brazilian patients with different treatment outcomes.

J Proteomics 2014 Aug 27;108:198-208. Epub 2014 May 27.

Laboratório de Leishmanioses, Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, ES, Brazil. Electronic address:

Unlabelled: The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial.

Biological Significance: Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.
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http://dx.doi.org/10.1016/j.jprot.2014.05.010DOI Listing
August 2014

Local inflammatory response induced by scorpionfish Scorpaena plumieri venom in mice.

Toxicon 2012 Jul 20;60(1):4-11. Epub 2012 Mar 20.

Departamento de Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Av. Marechal Campos 1468, Maruípe, 29043-900 Vitória/ES, Brazil.

The Scorpaena plumieri fish venom induces a severe pain and edema, observed both clinically and experimentally. In order to understand more about the envenomation syndrome, the present study characterized experimentally the local acute inflammatory response induced by S. plumieri venom (SpV) in a mouse model of tissue injury. Our results demonstrated that the local inflammatory response provoked after 2 h of SpV injection in footpad of mice is characterized by release of pivotal pro-inflammatory mediators (TNF, IL-6 and MCP-1). These mediators could be associated with histopathological changes observed into paw tissue, characterized by cellular infiltration, mainly neutrophils. Additionally, an investigation of edema formation pathways involved in inflammatory response was performed. SpV-induced edema was reduced significantly by previous administration of aprotinin or icatibant (HOE-140). However, the pre-treatment with diclofenac sodium and promethazine had less effect on this response. These results demonstrate that the kallikrein-kinin system (KKS) plays a major role in the edema formation. Despite the whole venom hydrolyzed the kallikrein synthetic substrate S-2302 (Pro-Phe-Arg-pNA), its main pro-inflammatory fraction was devoid of kininogenase activity. Our results demonstrate that SpV evokes a complex inflammatory reaction stimulating a secretion of TNF, IL-6, MCP-1 and leukocytes recruitment at the site of venom injection. In addition provide clear evidence of the involvement of the KKS in inflammatory response induced by S. plumieri venom.
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http://dx.doi.org/10.1016/j.toxicon.2012.03.008DOI Listing
July 2012

Stonefish antivenom neutralises the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom.

Toxicon 2011 Jun 13;57(7-8):992-9. Epub 2011 Apr 13.

Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, Brazil.

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynisStonefish Antivenom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5-100 μg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 μg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 μg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ∼98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.
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http://dx.doi.org/10.1016/j.toxicon.2011.04.001DOI Listing
June 2011
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