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Leishmaniases belong to the inglorious group of neglected tropical diseases, presenting different degrees of manifestations severity. It is caused by the transmission of more than 20 species of parasites of the Leishmania genus. Nevertheless, the disease remains on the priority list for developing new treatments, since it affects millions in a vast geographical area, especially low-income people. Molecular biology studies are pioneers in parasitic research with the aim of discovering potential targets for drug development. Among them are the telomeres, DNA–protein structures that play an important role in the long term in cell cycle/survival. Telomeres are the physical ends of eukaryotic chromosomes. Due to their multiple interactions with different proteins that confer a likewise complex dynamic, they have emerged as objects of interest in many medical studies, including studies on leishmaniases. This review aims to gather information and elucidate what we know about the phenomena behind Leishmania spp. telomere maintenance and how it impacts the parasite’s cell cycle.

Made available in DSpace on 2022-04-28T19:47:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-28 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Leishmania developmental cycle comprises three main life forms in two hosts, indicating that the parasite is continually challenged due to drastic environmental changes. The disruption of this cycle is critical for discovering new therapies to eradicate leishmaniasis, a neglected disease that affects millions worldwide. Telomeres, the physical ends of chromosomes, maintain genome stability and cell proliferation and are potential antiparasitic drug targets. Therefore, understanding how telomere length is regulated during parasite development is vital. Here, we show that telomeres form clusters spread in the nucleoplasm of the three parasite life forms. We also observed that amastigotes telomeres are shorter than metacyclic and procyclic promastigotes and that in parasites with continuous in vitro passages, telomere length increases over time. These observed differences in telomere length among parasite’s life stages were not due to lack/inhibition of telomerase since enzyme activity was detected in all parasite life stages, although the catalysis was temperature-dependent. These data led us to test if, similar to other eukaryotes, parasite telomere length maintenance could be regulated by Hsp83, the ortholog of Hsp90 in trypanosomatids, and Leishmania (LHsp90). Parasites were then treated with the Hsp90 inhibitor 17AAG. The results showed that 17AAG disturbed parasite growth, induced accumulation into G2/M phases, and telomere shortening in a time-dependent manner. It has also inhibited procyclic promastigote’s telomerase activity. Besides, LHsp90 interacts with the telomerase TERT component as shown by immunoprecipitation, strongly suggesting a new role for LHsp90 as a parasite telomerase component involved in controlling telomere length maintenance and parasite life span. Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP) Faculdade Brazileira Multivix Hemocentro da Faculdade de Medicina de Ribeirão Preto Universidade of São Paulo São Carlos Institute of Chemistry University of São Paulo Department of Chemical and Biological Sciences Institute of Biosciences São Paulo State University (UNESP)

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

Made available in DSpace on 2021-06-25T11:56:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Foreign Affairs, Trade, and Development Canada (DFATD) grant Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) CIHR ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance. Univ Campinas UNICAMP, Inst Chem, BR-13083970 Campinas, SP, Brazil Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil Univ Toronto, Dept Biochem, Toronto, ON M5G 1M1, Canada Univ Toronto, Dept Chem, Toronto, ON M5S 3H6, Canada Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil FAPESP: 2012/50161-8 FAPESP: 2017/261315 FAPESP: 2013/10939-2 FAPESP: 2015/13521-4 FAPESP: 2014/25967-4 FAPESP: 2019/11496-3 CAPES: 99999.004913/2015-09 CIHR: PJT-173491

Made available in DSpace on 2021-06-25T11:52:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) CIHR Heat shock proteins (Hsps) are involved in several important aspects of the cell proteostasis. Hsp90 interacts with at least a tenth of the cell proteome helping a large number of proteins to fold correctly. Hsp90 function is modulated by several co-chaperones having TPR (tetratricopeptide repeat) domains that allow for interaction with the C-terminal MEEVD motif of the chaperone. Another important chaperone, Hsp70, has a C-terminal EEVD motif that binds to TPR. Leishmania is a protozoan that causes leishmaniasis, a neglected disease in humans and other animals. There is still no effective treatment for leishmaniasis, however the study of structure and function of the proteins of the parasite may generate potential targets for future therapeutic intervention studies. In this work, the genome of Leishmania major was searched for a novel TPR-domain gene, which is conserved in Leishmania. The recombinant protein, LmTPR, was produced in pure and folded state and was characterized by biophysical tools as a monomer with an elongated conformation. Studies in Leishmania major were also preformed to complement these in vitro experiments. Splice Leader RNA-seq analysis and Western blot indicated that the protein was expressed in all developmental stages of the parasite. Binding assays confirmed that both Hsp90 and Hsp70 bind specifically to LmTPR. Finally, sequence and structural predictions indicated a C terminal region as a RPAP3 domain. Altogether, this study identified a novel TPR-domain co-chaperone of Hsp90 that is conserved and expressed in all developmental stages of Leishmania major. (C) 2021 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved. Univ Campinas UNICAMP, Inst Chem, BR-13083970 Campinas, SP, Brazil Natl Inst Sci & Technol Struct Biol & Bioimage IN, Rio De Janeiro, RJ, Brazil Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil Univ Sao Paulo, Sao Carlos Inst Chem, Sao Carlos, SP, Brazil Univ Toronto, Dept Biochem, Toronto, ON M5G 1M1, Canada Univ Toronto, Dept Chem, Toronto, ON M5S 3H6, Canada Sao Paulo State Univ, Biosci Inst, Dept Chem & Biol Sci, BR-18618689 Botucatu, SP, Brazil FAPESP: 2012/50161-8 FAPESP: 2017/26131-5 FAPESP: 2018/04375-2 FAPESP: 2014/25967-4 FAPESP: 2019/11496-3 CAPES: 99999.004913/2015-09 CIHR: PJT-173491

Telomeres are specialized sequences at the end of linear chromosomes. Its conserved structure and function among eukaryotic cells suggest important evolutionary functions. Telomere dynamics play major roles in chromosomal integrity, stability, cellular replication and aging, performing crucial genome protective functions. In the majority of somatic cells, telomeres shorten in each round of cell replication. Whereas short telomeres are a trigger for apoptosis, accelerated attrition is a hallmark of aging, present in senescent and tumoral cells. Compensation for erosion in germinal and progenitor cells is accomplished by the telomerase enzyme, composed of a catalytic (TERT) and a RNA template (TR) subunits. Telomerase activity is tightly controlled with reactivation central for tumoral transformation. Clinical evidence suggests that telomeres’ shortening that causally contributes to the establishment of specific progeria syndromes phenotypes are telomeropathies. In other cases, aging is accompanied by an abrupt telomere shortening in the context of chronic diseases, proposing telomeres length as an important biological pace marker for progression of various pathologies and aging. Because cancer cells reactivate TERT to compensate for telomeric loss, in the last decades, telomerase and the telomeres biology field have been subject to intense research in search for therapeutical targets for cancer. This chapter mainly focuses on basic telomere description and synthesis accomplished by the reverse transcriptase telomerase and its regulation. A final section addresses the understanding of telomeres in health and its contribution to cancer with therapeutic potentials for targeted inhibition.

RPA is a conserved heterotrimeric complex and the major single-stranded DNA (ssDNA)-binding protein heterotrimeric complex, which in eukaryotes is formed by the RPA-1, RPA-2, and RPA-3 subunits. The main structural feature of RPA is the presence of the oligonucleotide/oligosaccharide-binding fold (OB-fold) domains, responsible for ssDNA binding and protein:protein interactions. Among the RPA subunits, RPA-1 bears three of the four OB folds involved with RPA-ssDNA binding, although in some organisms RPA-2 can also bind ssDNA. The OB-fold domains are also present in telomere end-binding proteins (TEBP), essential for chromosome end protection. RPA-1 from Leishmania sp., as well as RPA-1 from trypanosomatids, a group of early-divergent protozoa, shows some structural differences compared to higher eukaryote RPA-1. Also, RPA-1 from Leishmania sp., similar to TEBPs, may exert telomeric protective functions. Remarkably, different pieces of evidence have pointed out that trypanosomatids may not have OB fold-containing TEBPs. Moreover, recent data indicate that trypanosomatid RPA-1 may be considered a TEBP since it shares with TEBPs conserved functional and structural features. However, it is still unknown whether the RPA-1 protective telomeric role is exclusive to trypanosomatids or is also present in other primitive eukaryotes. Here, we describe a protocol to obtain highly purified and biologically active Leishmania amazonensis recombinant RPA-1, and to perform molecular modeling and molecular dynamics simulations methods which could be probably applied to functional and structural studies of homologous proteins in other primitive eukaryotes.

© 2021 Elsevier B.V. All rights reserved., Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3′-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5′ upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRAR loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite., This work was supported by São Paulo Research Foundation (FAPESP, Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil, http://www.fapesp.br/), grant 2018/04375-2 to MINC and grant 2018/23302-6 to SG. The National Council for Scientific and Technological Development in Brazil, CNPq (grant 405581/2018-1), http://www.cnpq.br/ also supports SG. Morea, E.G.O. is a postdoctoral fellow from FAPESP (grant 2019/11496-3) and, MINC is a PQ-CNPq fellow (grant 302433/2019-8). The funders had no role in study design, data collec-tion, analysis, decision to publish, or manuscript preparation.

Telomere length maintenance is important for genome stability and cell division. In most eukaryotes, telomeres are maintained by the telomerase ribonucleoprotein (RNP) complex, minimally composed of the Telomerase Reverse Transcriptase (TERT) and the telomerase RNA (TER) components. In addition to TERT and TER, other protein subunits are part of the complex and are involved in telomerase regulation, assembly, disassembly, and degradation. Among them are some molecular chaperones such as Hsp90 and its co-chaperone p23 which are found associated with the telomerase RNP complex in humans, yeast and probably in protozoa. Hsp90 and p23 are necessary for the telomerase RNP assembly and enzyme activity. In budding yeast, the Hsp90 homolog (Hsp82) is also responsible for the association and dissociation of telomerase from the telomeric DNA by its direct interaction with a telomere end-binding protein (Cdc13), responsible for regulating telomerase access to telomeres. In addition, AAA+ ATPases, such as Pontin and Reptin, which are also considered chaperone- like proteins, associate with the human telomerase complex by the direct interaction of Pontin with TERT and dyskerin. They are probably responsible for telomerase RNP assembly since their depletion impairs the accumulation of the complex. Moreover, various RNA chaperones, are also pivotal in the assembly and migration of the mature telomerase complex and complex intermediates. In this review, we will focus on the importance of molecular chaperones for telomerase RNP biogenesis and how they impact telomere length maintenance and cellular homeostasis.

Made available in DSpace on 2020-12-12T02:01:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Centre National de la Recherche Scientifique Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Institut National de la Santé et de la Recherche Médicale Muséum National d'Histoire Naturelle Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Background: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. Methods and results: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3′-5′ Exonuclease I digestion, and unfold telomeric G-quadruplex. Conclusions: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes. Department of Biophysics and Pharmacology Biosciences Institute São Paulo State University (UNESP) – Botucatu Laboratoire de Biologie et Pharmacologie Appliquée École Normale Supérieure Paris-Saclay Department of Chemical and Biological Sciences São Paulo State University (UNESP) – Botucatu MNHN CNRS UMR 7196 INSERM U1154, 43 rue Cuvier Sorbonne Université UFR927, 4 place Jussieu Department of Biophysics and Pharmacology Biosciences Institute São Paulo State University (UNESP) – Botucatu Department of Chemical and Biological Sciences São Paulo State University (UNESP) – Botucatu CAPES: 171895/2018-01 FAPESP: 2015/17286-0 FAPESP: 2015/18641-5 FAPESP: 2018/0395-6 FAPESP: 2018/04375-2 FAPESP: 2019/11496-3 CNPq: 305300/2006-7 CAPES: 88881

Background Leishmania spp. telomeres are composed of 5′-TTAGGG-3′ repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Methods and results Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3′G-overhang, and DNA binding is calcium dependent. Conclusions We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. General significance LCalA is the first reported calmodulin that binds in vivo telomeric DNA.

Made available in DSpace on 2020-12-12T01:51:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In the past decade, research efforts were made to identify molecular biomarkers useful as therapeutic targets in Non-Small Cell Lung Cancer (NSCLC), the most frequent type of lung carcinoma. NSCLC presents different histological subtypes being the most prevalent LUSC (Lung Squamous Cell Cancer) and LUAD (Lung Adenocarcinoma), and only a subset of LUAD patients' present tumors expressing known targetable genetic alterations. Telomeres and its components, including telomerase, the enzyme that replenishes telomeres, have been considered potential cancer biomarkers due to their crucial role in cell proliferation and genome stability. Our study aims to quantify expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. We first assessed the transcriptome (RNA-Seq) data of NSCLC patients from The Cancer Genome Atlas (TCGA) and then we tested the expression of telomere-associated genes and telomeric ncRNAs (TERC, telomerase RNA component, and TERRA, telomere repeat-containing RNA) in Brazilian NCSLC patient samples by quantitative RT-PCR, using matched normal adjacent tissue samples as the control. We also estimated the mean size of terminal restriction fragments (TRF) of some Brazilian NSCLC patients using telomeric Southern blot. The TCGA analysis identified alterations in the expression profile of TERT and telomere damage repair genes, mainly in the LUSC subtype. The study of Brazilian NSCLC samples by RT-qPCR showed that LUSC and LUAD express high amounts of TERT and that although the mean TRF size of tumor samples was shorter compared to normal cells, telomeres in NSCLC are probably maintained by telomerase. Also, the expression analysis of Brazilian NSCLC samples identified statistically significant alterations in the expression of genes involved with telomere damage repair, as well as in TERC and TERRA, mainly in the LUSC subtype. We, therefore, concluded that telomere maintenance genes are significantly deregulated in NSCLC, representing potential biomarkers in the LUSC subtype. Genetics Dept. Biosciences Institute Sao Paulo State University (UNESP) Biostatics Dept. Biosciences Institute Sao Paulo State University (UNESP) Faculty of Veterinary Medicine and Animal Science Faculty of Medicine Sao Paulo State University (UNESP) Department of Surgery and Orthopedics Faculty of Medicine Sao Paulo State University (UNESP) Department of Pathology Faculty of Medicine Sao Paulo State University (UNESP) Leeds Omics University of Leeds Experimental Research Unity (UNIPEX) Faculty of Medicine Sao Paulo State University (UNESP) Genetics Dept. Biosciences Institute Sao Paulo State University (UNESP) Biostatics Dept. Biosciences Institute Sao Paulo State University (UNESP) Faculty of Veterinary Medicine and Animal Science Faculty of Medicine Sao Paulo State University (UNESP) Department of Surgery and Orthopedics Faculty of Medicine Sao Paulo State University (UNESP) Department of Pathology Faculty of Medicine Sao Paulo State University (UNESP) Experimental Research Unity (UNIPEX) Faculty of Medicine Sao Paulo State University (UNESP) FAPESP: 2012/50161-8 FAPESP: 2015/18641-5 FAPESP: 2016/06936- 6

Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD + -dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi , the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi , TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC 50 ) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a heterotrimeric complex formed by RPA-1, RPA-2 and RPA-3. RPA is a fundamental player in replication, repair, recombination and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruzi RPA presents canonical functions being involved with DNA replication and DNA damage response. Here we found by FISH/IF assays that T. cruzi RPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA-1. Telomeric DNA induces different secondary structural modifications on RPA-1 in comparison with other types of DNA. In addition, RPA-1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region. This article is protected by copyright. All rights reserved.

Leishmaniasis is a spectrum of diseases caused by parasites of the genus Leishmania that affects millions of people around the world. During infection, the parasites use different strategies to survive the host's defenses, including overcoming exposure to reactive oxidant species (ROS), responsible for causing damage to lipids, proteins and DNA. This damage especially affects telomeres, which frequently results in genome instability, senescence and cell death. Telomeres are the physical ends of the chromosomes composed of repetitive DNA coupled with proteins, whose function is to protect the chromosomes termini and avoid end-fusion and nucleolytic degradation. In this work, we induced acute oxidative stress in promastigote forms of Leishmania amazonensis by treating parasites with 2mM hydrogen peroxide (H2O2) for 1h, which was able to increase intracellular ROS levels. In addition, oxidative stress induced DNA damage, as confirmed by 8-oxodGuo quantification and TUNEL assays and the dissociation of LaRPA-1 from the 3' G-overhang, leading to telomere shortening. Moreover, LaRPA-1 was observed to interact with newly formed C-rich single-stranded telomeric DNA, probably as a consequence of the DNA damage response. Nonetheless, acute oxidative stress caused the death of some of the L. amazonensis population and induced cell cycle arrest at the G2/M phase in survivor parasites, which were able to continue proliferating and replicating DNA and became more resistant to oxidative stress. Taken together, these results suggest that adaptation occurs through the selection of the fittest parasites in terms of repairing oxidative DNA damage at telomeres and maintaining genome stability in a stressful environment.

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi., Author Summary Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, this parasite alternates between proliferative/non-infective forms and forms that are infective but not able to proliferate. Some stressors, such as acidic pH and starvation, trigger the transition from one form to another; however, many molecules involved in this response remain to be identified. Replication Protein A (RPA) is a single stranded DNA binding protein involved in many functions of DNA metabolism, such as DNA replication and repair. Although RPA is well characterized in yeast and mammalian cells, nothing is known about this heterotrimer in T. cruzi. Here, we demonstrated that T. cruzi RPA is involved in canonical functions of DNA metabolism. Accordingly, when we reduced the expression levels of subunit 2 of TcRPA (TcRPA-2) the growth of the replicative form of T. cruzi was compromised. Moreover, we observed that the impairment of cell growth is linked with the differentiation process because the reduction of the level of TcRPA-2 increased the capacity of the proliferative epimastigote form to differentiate into an infective metacyclic trypomastigote one. In conclusion, TcRPA has canonical functions in the ancient eukaryote T. cruzi and is also involved in the control of life cycle progression.

Telomeres are protein–DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmani amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2′-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.

Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.

The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5'-TTAGGG-3' telomeric repeats. Protein complexes that associate in vitro with these DNA sequences, Leishmania amazonensis G-strand telomeric protein (LaGT1-3), were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with double-stranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligonucleotides, or (c) after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3' G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as approximately 35 and approximately 52 kDa bands, respectively. The

Telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DNA double-strand breaks. With few exceptions, telomeres are composed primarily of repetitive DNA associated with proteins that interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in telomere maintenance and length regulation. In proliferative cells and unicellular organisms, telomeric DNA is replicated by the actions of telomerase, a specialized reverse transcriptase. In the absence of telomerase, some cells employ a recombination-based DNA replication pathway known as alternative lengthening of telomeres. However, mammalian somatic cells that naturally lack telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence. In another way, mutations or deletions of telomerase components can lead to inherited genetic disorders, and the depletion of telomeric proteins can elicit the action of distinct kinases-dependent DNA damage response, culminating in chromosomal abnormalities that are incompatible with life. In addition to the intricate network formed by the interrelationships among telomeric proteins, long noncoding RNAs that arise from subtelomeric regions, named telomeric repeat-containing RNA, are also implicated in telomerase regulation and telomere maintenance. The goal for the next years is to increase our knowledge about the mechanisms that regulate telomere homeostasis and the means by which their absence or defect can elicit telomere dysfunction, which generally results in gross genomic instability and genetic diseases.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major , and Leishmania tarentolae . Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. (1994) Science 266, 2011–2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928–5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei . Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

We report the molecular characterization of a middle repetitive DNA sequence, named C6, isolated from the Trypanosoma cruzi genome. C6 appears to be a composite repeated element since 3 subregions may be defined within it on the basis of sequence similarities with other T. cruzi genomic sequences. Sequences homologous to C6 are interspersed in the genome and can be mapped out on most chromosomal bands of different T. cruzi strains. The copy number of the C6 element is about 1000 per haploid genome. Given the species specificity and different genomic distribution of C6 homologous sequences among the T. cruzi strains the C6 element could be a useful probe for diagnosis and typing of parasites. C6 is a polymorphic marker with potential as a tool for physical mapping of the T. cruzi genome.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas’ disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain. © 1997 Elsevier Science B.V.

SUMMARYWe have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:28Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:37:31Z : No. of bitstreams: 1 2-s2.0-84877140924.pdf: 3952804 bytes, checksum: 0e428a0e5b772b18ef0debdf9dbd745e (MD5) Made available in DSpace on 2014-05-27T11:29:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-07 Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al. Departamento de Microbiologia, Imunologia e Parasitologia Escola Paulista de Medicina Universidade Federal de São Paulo, São Paulo, São Paulo Skirball Institute of Biomolecular Medicine New York University Cancer Center New York University School of Medicine, New York, NY Departamento de Genética Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, São Paulo Departamento de Bioquímica e Imunologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais Campus Centro-Oeste Dona Lindu Universidade Federal de São João del-Rey, Divinópolis, Minas Gerais Departamento de Genética Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, São Paulo

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

We have cloned and sequenced a cDNA clone coding for a metacyclic trypomastigote-specific surface glycoprotein with a molecular mass of 82 kDa (MTS-gp82). By immunoblotting and immunoprecipitation, antibodies against the recombinant protein recognized an 82-kDa protein of metacyclic trypomastigotes, without any detectable reaction towards amastigotes, epimastigotes or tissue culture-derived trypomastigotes. The insert of the MTS-gp82 cDNA clone strongly hybridized with a single 2.2-kb metacyclic trypomastigote mRNA, suggesting that the steady-state levels of mRNAs for MTS-gp82 are developmentally regulated. MTS-gp82 is encoded by a multigene family whose members are distributed in several chromosomes. Sequence analysis revealed 40-56% identity at amino acid level between MTS-gp82 and members of Trypanosoma cruzi gp85/sialidase family (TSA-1, Tt34c1, SA85-1.1). MTS-gp82 showed several amino acid motifs that are characteristic of gp85/sialidase family, such as the Asp box (SxDxGxTW), the subterminal (VTVxNVFLYNR) motif and the putative GPI-anchor sequence. On the basis of its structural features, the MTS-gp82 gene could be included in the T. cruzi gp85/sialidase family, but constituting a distinct group which is preferentially expressed in metacyclic trypomastigotes.

SUMMARYSirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue fromLeishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin‑affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylatedin vivo.

Made available in DSpace on 2022-04-28T22:02:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-01 The present review intends to summarize the, yet preliminary, but very important emerging data underlining the functions exerted by the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1 on protein homeostasis. The main focus of the discussion is the cooperation between SIRT1 and the heat shock factor 1 (HSF1) responsible for activating the transcription of molecular chaperones, the protein-protective factors that resolve damaged/misfolded and aggregated proteins generated by heat stress or metabolism. SIRT1, a mammalian ortholog of the yeast silent information regulator 2, is a stress activated protein deacetylase that contributes to life-span extension by regulating different cell survival pathways, including replicative senescence, inflammation and resistance to hypoxic and heat stress. Phosphorylation is the major mechanism controlling the level and function of SIRT1 required for normal cell cycle progression and cell survival under stress conditions. Phosphorylated SIRT1 deacetylates and coactivates different substrates, including HSF1. Deacetylated HSF1 binds to the heat shock promoter element found upstream of genes encoding molecular chaperones. Overexpression of SIRT1 in cultured cells also helps them to survive exposure to heat stress. Conversely, its down-regulation accelerates the attenuation of the heat shock response promoting the release of HSF1 from its cognate promoter element. Very recently, in a mouse model for Alzheimer's disease, SIRT1 deacetylase activity was also found activating the transcription of α-secretase, the enzyme responsible for inhibiting the formation of aggregates of neuronal β-amyloid plaques. How SIRT1 activity protects cells from the deleterious effects of damaged/misfolded proteins and the implication of these findings on age-related pathologies are discussed. © 2011 Bentham Science Publishers Ltd. Depto. de Genetica IBB UNESP, Rua 02, s/no., Botucatu, 18618-970, Rubiao Junior, Botucatu, Sao Paulo Depto. de Genetica IBB UNESP, Rua 02, s/no., Botucatu, 18618-970, Rubiao Junior, Botucatu, Sao Paulo

Made available in DSpace on 2013-08-28T14:08:49Z (GMT). No. of bitstreams: 1 WOS000279927400001.pdf: 1602412 bytes, checksum: 525358379230aad20fa41f7f8ae664f0 (MD5) Made available in DSpace on 2013-09-30T18:37:53Z (GMT). No. of bitstreams: 2 WOS000279927400001.pdf: 1602412 bytes, checksum: 525358379230aad20fa41f7f8ae664f0 (MD5) WOS000279927400001.pdf.txt: 42185 bytes, checksum: fad5da0aa82e434f4180e7977ba95352 (MD5) Previous issue date: 2010-05-07 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:27Z No. of bitstreams: 2 WOS000279927400001.pdf: 1602412 bytes, checksum: 525358379230aad20fa41f7f8ae664f0 (MD5) WOS000279927400001.pdf.txt: 42185 bytes, checksum: fad5da0aa82e434f4180e7977ba95352 (MD5) Made available in DSpace on 2014-05-20T13:50:27Z (GMT). No. of bitstreams: 2 WOS000279927400001.pdf: 1602412 bytes, checksum: 525358379230aad20fa41f7f8ae664f0 (MD5) WOS000279927400001.pdf.txt: 42185 bytes, checksum: fad5da0aa82e434f4180e7977ba95352 (MD5) Previous issue date: 2010-05-07 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2.Results: LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo.Conclusion: The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein. Univ Estadual Paulista, UNESP, Telomeres Lab, Dept Genet,Biosci Inst, Botucatu, SP, Brazil Inst Pasteur Korea, Ctr Neglected Dis Drug Discovery, Gyeonggi Do, South Korea Univ Estadual Paulista, UNESP, Telomeres Lab, Dept Genet,Biosci Inst, Botucatu, SP, Brazil FAPESP: 06/58175-7 CNPq: 481850/2008

Departamento de Micro, Imuno e Parasitologia, Escola Paulista de Medicina, Unifesp, Rua Botucatu 862, 04023-062 Sao Paulo, SP, Brasil *Unidad de Parasitologia, Universidad de Antofagasta, Av. Coloso s/n, Casilla 170,Antofagasta, Chile **Instituto de Biologia Experimental, Universidad Central de Venezuela, Aptado 47525,Caracas 1041-A, VenezuelaKey words: Trypanosoma cruzi - metacyclic trypomastigotes - surface glycoprotein genes - genomic organization- transcription

Made available in DSpace on 2013-08-28T14:09:27Z (GMT). No. of bitstreams: 1 WOS000263248800003.pdf: 493620 bytes, checksum: da3021554a79b14560744cae801be0d3 (MD5) Made available in DSpace on 2013-09-30T18:37:52Z (GMT). No. of bitstreams: 2 WOS000263248800003.pdf: 493620 bytes, checksum: da3021554a79b14560744cae801be0d3 (MD5) WOS000263248800003.pdf.txt: 33384 bytes, checksum: a5b9e409f72a067405e21f8a58cb8a44 (MD5) Previous issue date: 2008-06-11 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:27Z No. of bitstreams: 2 WOS000263248800003.pdf: 493620 bytes, checksum: da3021554a79b14560744cae801be0d3 (MD5) WOS000263248800003.pdf.txt: 33384 bytes, checksum: a5b9e409f72a067405e21f8a58cb8a44 (MD5) Made available in DSpace on 2014-05-20T13:50:27Z (GMT). No. of bitstreams: 2 WOS000263248800003.pdf: 493620 bytes, checksum: da3021554a79b14560744cae801be0d3 (MD5) WOS000263248800003.pdf.txt: 33384 bytes, checksum: a5b9e409f72a067405e21f8a58cb8a44 (MD5) Previous issue date: 2008-06-11 Institut Pasteur Korea Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed. Inst Pasteur Korea, Syst Biol Pathogens Grp, Seoul, South Korea Inst Pasteur Korea, Image Mining Grp, Seoul, South Korea Univ Fed São Paulo, Dept Microbiol, Imunol Parasitol, São Paulo, Brazil Univ Estadual Paulista, Inst Biocien, Dept Genet, São Paulo, Brazil Univ Estadual Paulista, Inst Biocien, Dept Genet, São Paulo, Brazil

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP.

Made available in DSpace on 2015-10-21T13:11:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-16. Added 1 bitstream(s) on 2015-10-22T09:52:23Z : No. of bitstreams: 1 WOS000351183500197.pdf: 724905 bytes, checksum: e116e4368453deb0137ccac880d8e20e (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA. Inst Butantan, Lab Especial Ciclo Celular, Sao Paulo, Brazil Inst Butantan, Ctr Toxins Immune Response &Cell Signaling CeTIC, Sao Paulo, Brazil Univ Sao Paulo, Inst Ciencias Biomed, Dept Parasitol, Unit Drug Discovery, BR-05508 Sao Paulo, Brazil Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-01498 Sao Paulo, Brazil Univ Estadual Paulista Julio de Mesquita Filho UN, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil Univ Estadual Paulista Julio de Mesquita Filho UN, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil FAPESP: 2005/00154-1 FAPESP: 2013/07467-1 FAPESP: 2011/50631-1

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

The Leishmania amazonensis telomerase gene was cloned by a polymerase chain reaction-based strategy using primers designed from a Leishmania major sequence that shared similarities with conserved telomerase motifs. The genes from three other species were cloned for comparative purposes. A ClustalW multiple-sequence alignment demonstrated that the Leishmania telomerases show greater homology with each other than with the proteins of other kinetoplastids and eukaryotes. Characterization experiments indicated that the putative Leishmania telomerase gene was probably in single copy and located in the largest chromosomes. A single messenger ribonucleic acid transcript was found in promastigotes. Phylogenetic analysis suggested that Leishmania telomerase might represent a liaison between the oldest and the newest branches of telomerases.