Endoplasmic Reticulum Calcium Regulates the Retrotranslocation of Trypanosoma Cruzi Calreticulin to the Cytosol

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

Anticoagulant Activity of a Unique Sulfated Pyranosic (133) - ß - L - Arabinan through Direct Interaction with Thrombin

Fernández, Paula Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Química de Biomoléculas. Buenos Aires, Argentina.Quintana, Irene. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Hemostasia y Trombosis. Buenos Aires, Argentina.Cerezo, Alberto S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. Subsede del Centro de Investigación de Hidratos de Carbono (CIHIDECAR). Ciudad Universitaria, Buenos Aires, Argentina.Caramelo, Julio J. CONICET - Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA). Buenos Aires, Argentina.Pol Fachin, Laercio. Universidade Federal do Rio Grande do Sul. Faculdade de Farmácia. Programa de Pos-Graduação em Biologia Celular e Molecular. Centro de Biotecnologia. Rio Grande do Sul, Brasil.Verli, Hugo. Universidade Federal do Rio Grande do Sul.Centro de Biotecnologia. Programa de Pos-Graduação em Biologia Celular e Molecular. Rio Grande do Sul, Brasil.Estevez, José Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias (IFIByNE). Buenos Aires, Argentina.Ciancia, Marina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Química de Biomoléculas. Buenos Aires, Argentina.223–233Background: Many seaweed polysaccharides have anticoagulant activity, but the mechanism of action was elucidated in a few cases. Results: A highly sulfated pyranosic ß-arabinan exerts its activity through direct and indirect inhibition of thrombin. Conclusion: The structure and mechanism of action of the arabinan are different from those found for other polysaccharides. Significance: This arabinan could be an alternative anticoagulant in certain specific cases

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 A resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin “B” delivery subunit would bind to target membranes, and then the MACPF “A” toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Estudios Inmunológicos y Fisiopatológico

Structural coalescence underlies the aggregation propensity of a β-barrel protein motif.

A clear understanding of the structural foundations underlying protein aggregation is an elusive goal of central biomedical importance. A step toward this aim is exemplified by the β-barrel motif represented by the intestinal fatty acid binding protein (IFABP) and two abridged all-β sheet forms (Δ98Δ and Δ78Δ). At odds with the established notion that a perturbation of the native fold should necessarily favor a buildup of intermediate forms with an enhanced tendency to aggregate, the intrinsic stability (ΔG°H2O) of these proteins does not bear a straightforward correlation with their trifluoroethanol (TFE)-induced aggregation propensity. In view of this fact, we found it more insightful to delve into the connection between structure and stability under sub-aggregating conditions (10% TFE). In the absence of the co-solvent, the abridged variants display a common native-like region decorated with a disordered C-terminal stretch. Upon TFE addition, an increase in secondary structure content is observed, assimilating them to the parent protein. In this sense, TFE perturbs a common native like region while exerting a global compaction effect. Importantly, in all cases, fatty acid binding function is preserved. Interestingly, energetic as well as structural diversity in aqueous solution evolves into a common conformational ensemble more akin in stability. These facts reconcile apparent paradoxical findings related to stability and rates of aggregation. This scenario likely mimics the accrual of aggregation-prone species in the population, an early critical event for the development of fibrillation

Size-exclusion chromatography.

<p>Size-exclusion chromatography of IFABP (solid line), Δ98Δ (dashed line) and Δ78Δ (dotted line). Proteins were sampled onto a Superdex-75 column and eluted at 10% v/v TFE in buffer PN8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#sec002" target="_blank">Materials and methods</a>). Arrows indicate the elution volumes in the absence of TFE.</p

Urea-induced unfolding transitions.

<p>Evolution of the fluorescence emission (integrated intensity) as a function of urea concentration (Panel A) for IFABP (■, □), Δ98Δ (●,○) and Δ78Δ (▲,△). Experiments were carried out in the absence (closed symbols) or in the presence of 10% v/v TFE (open symbols). Fitted curves of the native molar fraction of IFABP (solid line), Δ98Δ (dashed line) or Δ78Δ (dotted line) are plotted at 0 (panel B) and 10% v/v TFE (panel C). Notice that for the dimeric construct Δ78Δ -that has been described to dissociate and unfold concomitantly- the free energies (ΔG° values) of the overall process (expressed in protomer equivalents, see reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170607#pone.0170607.ref007" target="_blank">7</a>]) are tabulated.</p