RESUMO
N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.
Assuntos
Hexosiltransferases , Proteínas de Membrana , Processamento de Proteína Pós-Traducional , Humanos , Glicosilação , Células HEK293 , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Estabilidade Proteica , Polissacarídeos/metabolismoRESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoan parasite that infects phagocytic and non-phagocytic mammalian cells. At early stages of infection, trypomastigotes, the infective forms of this parasite, localize in a vesicular compartment called the T. cruzi parasitophorous vacuole until the exit of parasites to the host cell cytoplasm where continue their infective cycle. Rab proteins participate in the membrane traffic's molecular machinery, functioning as central regulators of vesicle recognition and transport. In previous work, we demonstrated that endocytic Rabs are key factors of the T. cruzi infection process in non-phagocytic cells, regulating the formation and the maturation of the vacuole. In this work, we identified and characterized other molecular components of the vesicular transport pathways and their participation in the T. cruzi infection. We found that Rab9a and Rab32, two regulators of the endocytic and autophagic pathways, were actively recruited to the T. cruzi vacuoles and favored the late stages of the infective process. The recruitment was specific and dependent on T. cruzi protein synthesis. Interestingly, Rab32 association depends on the presence of Rab9a in the vacuolar membrane, while the inhibition of the cysteine-protease cruzipain, a T. cruzi virulence factor, significantly decreases both Rab9a and Rab32 association with the vacuole. In summary, this work showed for the first time that specific molecules produced and secreted by the parasite can subvert intracellular components of host cells to benefit the infection. These new data shed light on the complex map of interactions between T. cruzi and the host cell and introduce concepts that can be useful in finding new forms of intervention against this parasite in the future.
RESUMO
T. cruzi, the causal agent of Chagas disease, is a parasite able to infect different types of host cells and to persist chronically in the tissues of human and animal hosts. These qualities and the lack of an effective treatment for the chronic stage of the disease have contributed to the durability and the spread of the disease around the world. There is an urgent necessity to find new therapies for Chagas disease. Drug repurposing is a promising and cost-saving strategy for finding new drugs for different illnesses. In this work we describe the effect of carvedilol on T. cruzi. This compound, selected by virtual screening, increased the accumulation of immature autophagosomes characterized by lower acidity and hydrolytic properties. As a consequence of this action, the survival of trypomastigotes and the replication of epimastigotes and amastigotes were impaired, resulting in a significant reduction of infection and parasite load. Furthermore, carvedilol reduced the whole-body parasite burden peak in infected mice. In summary, in this work we present a repurposed drug with a significant in vitro and in vivo activity against T. cruzi. These data in addition to other pharmacological properties make carvedilol an attractive lead for Chagas disease treatment.
Assuntos
Parasitos , Trypanosoma cruzi , Animais , Autofagia , Carvedilol/farmacologia , Reposicionamento de Medicamentos , CamundongosRESUMO
Cruzipain, the major cysteine protease of the pathogenic protozoa Trypanosoma cruzi, is an important virulence factor that plays a key role in the parasite nutrition, differentiation and host cell infection. Cruzipain is synthesized as a zymogen, matured, and delivered to reservosomes. These organelles that store proteins and lipids ingested by endocytosis undergo a dramatic decrease in number during the metacyclogenesis of T. cruzi. Autophagy is a process that digests the own cell components to supply energy under starvation or different stress situations. This pathway is important during cell growth, differentiation and death. Previously, we showed that the autophagy pathway of T. cruzi is induced during metacyclogenesis. This work aimed to evaluate the participation of macroautophagy/autophagy in the distribution and function of reservosomes and cruzipain during this process. We found that parasite starvation promotes the cruzipain delivery to reservosomes. Enhanced autophagy increases acidity and hydrolytic activity in these compartments resulting in cruzipain enzymatic activation and self- processing. Inhibition of autophagy similarly impairs cruzipain traffic and activity than protease inhibitors, whereas mutant parasites that exhibit increased basal autophagy, also display increased cruzipain processing under control conditions. Further experiments showed that autophagy induced cruzipain activation and self-processing promote T. cruzi differentiation and host cell infection. These findings highlight the key role of T. cruzi autophagy in these processes and reveal a potential new target for Chagas disease therapy.Abbreviations: Baf: bafilomycin A1; CTE: C-terminal extension; Cz: cruzipain; IIF: indirect immunofluorescence; K777: vinyl sulfone with specific Cz inhibitory activity; Prot Inh: broad-spectrum protease inhibitor; Spa1: spautin-1; Wort: wortmannin.
Assuntos
Autofagia/fisiologia , Doença de Chagas/metabolismo , Organelas/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Cisteína Endopeptidases/isolamento & purificação , Endocitose/imunologia , Parasitos/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/metabolismoRESUMO
trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Trypanosoma cruzi/isolamento & purificação , Animais , Doença de Chagas/sangue , Doença de Chagas/parasitologia , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Neuraminidase/análise , Neuraminidase/genética , Neuraminidase/imunologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Fatores de Virulência/sangue , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
As a part of our project aimed at developing new safe chemotherapeutic agents against tropical diseases, a series of aryl derivatives of 2- and 3-aminoquinoline, some of them new compounds, was designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible for American trypanosomiasis (Chagas' disease), and Leishmania mexicana, the etiological agent of Leishmaniasis. Some of them showed a remarkable activity as parasite growth inhibitors. Fluorine-containing derivatives 11b and 11c were more than twice more potent than geneticin against intracellular promastigote form of Leishmania mexicana exhibiting both IC50 values of 41.9⯵M. The IC50 values corresponding to fluorine and chlorine derivatives 11b-d were in the same order than benznidazole against epimastigote form. These drugs are interesting examples of effective antiparasitic agents with outstanding potential not only as lead drugs but also to be used for further in vivo studies. In addition, the obtained compounds showed no toxicity in Vero cells, which makes them good candidates to control tropical diseases. Regarding the probable mode of action, assayed quinoline derivatives interacted with hemin, inhibiting its degradation and generating oxidative stress that is not counteracted by the antioxidant defense system of the parasite.
Assuntos
Aminoquinolinas/farmacologia , Tripanossomicidas/farmacologia , Aminoquinolinas/síntese química , Aminoquinolinas/química , Aminoquinolinas/toxicidade , Animais , Chlorocebus aethiops , Hemina/metabolismo , Leishmania mexicana/efeitos dos fármacos , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.
Assuntos
Células Eucarióticas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Seleção Genética , Animais , Células COS , Chlorocebus aethiops , Biologia Computacional/métodos , Retículo Endoplasmático/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Glicoproteínas/química , Glicosilação , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Despite affecting around 8 million people worldwide and representing an economic burden above $7 billion/ year, currently approved medications to treat Chagas disease are still limited to two drugs, nifurtimox and benznidazole, which were developed more than 40 years ago and present important efficacy and safety limitations. Drug repositioning (i.e. finding second or further therapeutic indications for known drugs) has raised considerable interest within the international drug development community. There are many explanations to the current interest on drug repositioning including the possibility to partially circumvent clinical trials and the consequent saving in time and resources. It has been suggested as a particular attractive approach for the development of novel therapeutics for neglected diseases, which are usually driven by public or non-profit organizations. Here we review current computer-guided approaches to drug repositioning and reports on drug repositioning stories oriented to Chagas disease, with a focus on computer-guided drug repositioning campaigns.
Assuntos
Doença de Chagas/tratamento farmacológico , Reposicionamento de Medicamentos , Tripanossomicidas/uso terapêutico , Benzofuranos/química , Benzofuranos/farmacologia , Benzofuranos/uso terapêutico , Biologia Computacional , Ensaios de Triagem em Larga Escala , Humanos , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.
Assuntos
Reposicionamento de Medicamentos/métodos , Tripanossomicidas/farmacologia , Animais , Clofazimina/metabolismo , Clofazimina/farmacologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Di-Hidropiridinas/metabolismo , Di-Hidropiridinas/farmacologia , Feminino , Masculino , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas de Protozoários , Saquinavir/metabolismo , Saquinavir/farmacologia , Tripanossomicidas/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.
Assuntos
Antiprotozoários/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Proteínas de Protozoários/química , Tiroxina/química , Antiprotozoários/farmacologia , Domínio Catalítico , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Ligação Proteica , Proteínas de Protozoários/metabolismo , Tiroxina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Most sterols, such as cholesterol and ergosterol, become functional only after the removal of the two methyl groups at C-4 from their biosynthetic precursors. Nevertheless, some findings suggest that 4,4-dimethyl sterols might be involved in specific physiological processes. In this paper we present the synthesis of a collection of analogues of 4,4-dimethyl sterols with a diamide side chain and a preliminary analysis of their in vitro activity on selected biological systems. The key step for the synthesis involves an Ugi condensation, a versatile multicomponent reaction. Some of the new compounds showed antifungal and cytotoxic activity.
Assuntos
Células Eucarióticas/efeitos dos fármacos , Esteróis/biossíntese , Animais , Chlorocebus aethiops , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Esteróis/química , Esteróis/farmacologia , Células VeroRESUMO
Cruzipain (Cz) is the major cystein protease of the protozoan Trypanosoma cruzi , etiological agent of Chagas disease. From a 163 compound data set, a 2D-classifier capable of identifying Cz inhibitors was obtained and applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. Fifty-four approved drugs were selected as candidates, four of which were acquired and tested on Cz and T. cruzi epimastigotes. Among them, the antiparkinsonian and antidiabetic drug bromocriptine and the antiarrhythmic amiodarone showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.
Assuntos
Amiodarona/farmacologia , Bromocriptina/farmacologia , Desenho Assistido por Computador , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Reposicionamento de Medicamentos/métodos , Proteínas de Protozoários , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/-) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/- epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/- parasites was observed. Moreover, TcCRT+/- parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system.
Assuntos
Calreticulina/genética , Calreticulina/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Animais , Sequência de Bases , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , DNA de Protozoário/genética , Deleção de Genes , Genes de Protozoários , Humanos , Insetos Vetores/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Regulação para CimaRESUMO
A series of hydroxyalkyl and acyloxyalkyl derivatives of 2- and 3-hydroxypyridine was synthesized and their biological activity was evaluated as growth inhibitors of protozoan Leishmania mexicana. Thirty novel compounds were obtained through a chemoenzymatic methodology in two reaction steps. The influence of various reaction parameters in the enzymatic step, such as enzyme source, acylating agent/substrate ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evaluated compounds showed a remarkable activity as Leishmania mexicana growth inhibitors, obtaining the best results with the acetylated derivatives. The advantages showed by the enzymatic methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare these derivatives of substituted pyridines with application as potential antiparasitic agents.
Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Lipase/metabolismo , Piridinas/farmacologia , Antiprotozoários/química , Antiprotozoários/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Leishmania mexicana/crescimento & desenvolvimento , Lipase/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Piridinas/química , Piridinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the ß subunit (GIIß) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIß MRH domain and that the N-terminal GIIß G2B domain is involved in the GIIα-GIIß interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIß MRH domains with a higher specificity for glycans with high mannose content.
Assuntos
Glicoproteínas/metabolismo , Manose/metabolismo , Schizosaccharomyces/enzimologia , alfa-Glucosidases/metabolismo , Sequência de Carboidratos , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Meia-Vida , Hexosiltransferases/metabolismo , Dados de Sequência Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , alfa-Glucosidases/genética , alfa-ManosidaseRESUMO
Proteins may adopt diverse conformations during their folding in vivo, ranging from extended chains when they emerge from the ribosome to compact intermediates near the end of the folding process. Accordingly, a variety of chaperones and folding assisting enzymes have evolved to deal with this diversity. Chaperone selection by a particular substrate depends on the structural features of its folding intermediates. In addition, this process may be modulated by competitive effects between chaperones. Here we address this issue by using TcrCATL as model substrate. TcrCATL is an abundant Trypanosoma cruzi lysosomal protease and it was the first identified endogenous UDP-Glc:glycoprotein glucosyltransferase (UGGT) substrate. We found that TcrCATL associated sequentially with BiP and calreticulin (CRT) during its folding process. Early, extended conformations were bound to BiP, while more advanced and compact folding intermediates associated to CRT. The interaction between TcrCATL and CRT was impeded by deletion of the UGGT-encoding gene but, similarly to what was observed in wild type cells, in mutant cells TcrCATL associated to BiP only when displaying extended conformations. The absence of TcrCATL-CRT interactions in UGGT null cells resulted in a drastic reduction of TcrCATL folding efficiency and triggered the aggregation of TcrCATL through intermolecular disulfide bonds. These observations show that BiP and CRT activities complement each other to supervise a complete and efficient TcrCATL folding process. The present report provides further evidence on the early evolutionary acquisition of the basic tenets of the N-glycan dependent quality control mechanism of glycoprotein folding.
Assuntos
Calreticulina/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de Proteína , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Modelos Biológicos , Modelos QuímicosRESUMO
For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.
Assuntos
Cálcio/metabolismo , Calreticulina/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Transporte BiológicoRESUMO
Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.
Assuntos
Cálcio/farmacologia , Calreticulina/química , Animais , Cromatografia em Gel , Dicroísmo Circular , Análise de Fourier , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , CoelhosRESUMO
Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose alpha subunit (GIIalpha) bears the glycosyl hydrolase active site, whereas its beta subunit (GIIbeta) role is controversial and has been reported to be involved in GIIalpha ER retention and folding. Here, we report that in the absence of GIIbeta, the catalytic subunit GIIalpha of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl alpha-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc(2)Man(9)GlcNAc(2) (G2M9) and Glc(1)Man(9)GlcNAc(2) (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIbeta and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIbeta modulates G2M9 and G1M9 trimming.
Assuntos
Polissacarídeos/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Domínio Catalítico , Glucosídeos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutagênese , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Ratos , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genética , Alinhamento de Sequência , alfa-Glucosidases/genéticaRESUMO
Calreticulin, a Ca(2+) chaperone, is found in many different locations in various eukaryotic cells, including lumen of the endoplasmic reticulum, the cell surface, perinuclear areas and cytosolic granules. In the present study, a polyclonal antibody against calreticulin was used for the immunocytochemical localisation of the protein in Trypanosoma cruzi. Labelling was observed in the endoplasmic reticulum, Golgi complex, reservosomes, flagellar pocket, cell surface, cytosol, nucleus and kinetoplast. Significant differences in labelling were observed among the three evolutive forms of the protozoan. The functional role of calreticulin in T. cruzi is discussed.