Plasmodium vivax shows a strict host tropism for reticulocytes. We identify transferrin receptor 1 (TfR1) as the receptor for P. vivax reticulocyte-binding protein 2b (PvRBP2b). The structure of the N-terminal domain of PvRBP2b involved in red blood cell binding was determined, elucidating the molecular basis for TfR1 recognition. TfR1 was validated as the biological target of PvRBP2b engagement by TfR1 expression knockdown analysis. TfR1 mutant cells deficient in PvRBP2b binding were refractory to invasion of P. vivax, but not to invasion of P. falciparum. Using Brazilian and Thai clinical isolates, we show that PvRBP2b monoclonal antibodies that inhibit reticulocyte binding also block P. vivax entry into reticulocytes. These data show that TfR1-PvRBP2b invasion pathway is critical for the recognition of reticulocytes during P. vivax invasion.
The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5-the essential invasion ligand in Plasmodium falciparum-its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase-and chymotrypsinresistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.parasite invasion | X-ray crystallography | SAXS | reticulocyte binding protein | malaria T he most widely distributed recurring malaria infections globally are caused by Plasmodium vivax, which accounts for 80-100 million malaria infections per year (1). The majority of clinical symptoms associated with malaria are due to blood stage infection (2). The merozoite forms of malaria parasites invade human erythrocytes through a multistep process that involves initial contact with the red blood cell, apical reorientation of the merozoite, and the formation of a tight junction that moves progressively toward the posterior end of the parasite until host cell membrane fusion is completed. These steps in invasion are dependent on specific interactions between parasite adhesins and their cognate erythrocyte receptors (reviewed in ref.3).P. vivax preferentially invades reticulocytes: i.e., immature red blood cells (4). The basis of host cell selectivity by merozoites from Plasmodium spp. seems to be mediated primarily by families of adhesin proteins. The two erythrocyte-binding protein families of P. vivax are called the Duffy-binding protein (PvDBP) and reticulocyte-binding protein (PvRBP) families (5). In laboratory-adapted P. vivax strains, there is only one PvDBP protein in P. vivax that binds to Duffy antigen receptor for chemokines (DARC) (6...
Amyloid deposits are proteinaceous extra-cellular aggregates associated with a diverse range of disease states. These deposits are composed predominantly of amyloid fibrils, the unbranched, -sheet rich structures that result from the misfolding and subsequent aggregation of many proteins. In addition, amyloid deposits contain a number of non-fibrillar components that interact with amyloid fibrils and are incorporated into the deposits in their native folded state. The influence of a number of the non-fibrillar components in amyloid-related diseases is well established; however, the mechanisms underlying these effects are poorly understood. Here we describe the effect of two of the most important non-fibrillar components, serum amyloid P component and apolipoprotein E, upon the solution behavior of amyloid fibrils in an in vitro model system. Using analytical ultracentrifugation, electron microscopy, and rheological measurements, we demonstrate that these non-fibrillar components cause soluble fibrils to condense into localized fibrillar aggregates with a greatly enhanced local density of fibril entanglements. These results suggest a possible mechanism for the observed role of non-fibrillar components as mediators of amyloid deposition and deposit stability.The self-association of proteins into amyloid fibrils and the further aggregation of these fibrils into insoluble deposits is implicated in a diverse range of diseases, including Alzheimer's and other neurodegenerative diseases (1), type 2 diabetes (2), and a number of systemic amyloidoses (3). Over twenty proteins are known to form amyloid fibrils in vivo, and the deposits formed by each of these proteins are associated with a distinct disease state (4). Despite the lack of any similarity in primary sequence or native structure among these precursors, the underlying structures and histological properties of the deposits are remarkably similar. The bulk of the amyloid deposit is the amyloid fibrils themselves: linear unbranched assemblies of the specific precursor protein with a core cross- structure (5). Deposits also contain a number of non-fibrillar components, the identity of which is essentially independent of the precursor protein comprising the amyloid fibrils. These non-fibrillar components include the protein serum amyloid P component (SAP), 1 apolipoprotein E (apoE), as well as other proteins, glycosaminoglycans, and proteoglycans.The ubiquity and specificity of the non-fibrillar components of amyloid deposits is such that radiolabeled SAP is used clinically as a quantitative tracer of amyloid deposits (3). Furthermore, findings that SAP stabilizes in vitro amyloid deposits from phagocytic and proteolytic degradation has prompted the design of inhibitors of the SAP-amyloid interaction. One such molecule is under clinical trial against systemic amyloidosis (6). Despite this growing clinical interest in the effects of nonfibrillar components of amyloid deposits, structural details of their interactions with amyloid fibrils and deposits are entirely l...
The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-β1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.
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