Membrane protein misassembly in disease

Helix-helix interactions play a central role in the folding and assembly of integral α-helical membrane proteins and are fundamentally dictated by the amino acid sequence of the TM domain. It is not surprising then that missense mutations that target these residues are often linked to disease. In this review, we focus on the molecular mechanisms through which missense mutations lead to aberrant folding and/or assembly of these proteins, and then discuss pharmacological approaches that may potentially mitigate or reverse the negative effects of these mutations. Improving our understanding of how missense mutations affect the interactions between TM α-helices will increase our capability to develop effective therapeutic approaches to counter the misassembly of these proteins and, ultimately, disease. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin – Eh Klp5 – led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica . In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2–4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2–4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional.     

Autophosphorylation of Ser428 of EhC2PK plays a critical role in regulating erythrophagocytosis in the parasite Entamoeba histolytica

The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn(2+)-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.     

Proteomic analysis of Gal/GalNAc lectin-associated proteins in Entamoeba histolytica

Contact-dependent killing and phagocytosis of target cells by Entamoeba histolytica trophozoites is mediated by the galactose (Gal) and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. Previous work has suggested that this lectin functions as part of a signal transduction complex. To identify proteins that might be part of this complex, amebic trophozoites were bound to GalNAc-BSA-labeled magnetic beads and lysed. Bound proteins were eluted from the beads and analyzed by tandem mass spectrometry. Along with the Gal/GalNAc lectin subunits, several cytoskeletal proteins, potential signaling proteins, and a novel transmembrane protein, consistently purified with the GalNAc-BSA beads.     

Genomic and proteomic approaches highlight phagocytosis of living and apoptotic human cells by the parasite Entamoeba histolytica

Phagocytosis plays a major role during the invasive process of the human intestine by the pathogenic amoeba E. histolytica. This parasite is the etiologic agent causing amoebic dysentery, a worldwide disease causing 50 million of clinical cases leading to about 100,000 deaths annually. The invasive process is characterized by a local acute inflammation and the destruction of the intestinal tissue at the invasion site. The recent sequencing of the E. histolytica genome has opened the way to large-scale approaches to study parasite virulence such as processes involved in human cell phagocytosis. In particular, two different studies have recently described the phagosome proteome, providing new insights into the process of phagocytosis by this pathogenic protozoan. It has been previously described that E. histolytica induces apoptosis and phagocytosis of the human target cells. Induction of apoptosis by the trophozoites is thought to be involved in the close regulation of the inflammatory response occurring during infection. Little is known about the molecular mechanisms responsible for induction of apoptosis or in the recognition of apoptotic cells by E. histolytica. In this review, we comment on the recent data we obtained after isolation of the early phagosomes and the identification of its associated proteins. We focus on the surface molecules potentially involved in human cell recognition. In particular, we propose several parasite molecules, potentially involved in the induction of apoptosis and/or the phagocytosis of human apoptotic cells.     

Entamoeba histolytica: correlation between virulence and content of proteolytic enzymes

Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.     

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.     

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.     

Acid intracellular vesicles and the cytolysis of mammalian target cells by Entamoeba histolytica trophozoites

Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 +/- 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to greater than or equal to 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 microM). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4 degrees C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 micrograms/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 micrograms for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

A chemical approach towards understanding the mechanism and reversal of drug resistance in Plasmodium falciparum: is it viable?

Genetic and biochemical approaches to studies of drug resistance mechanisms in Plasmodium falciparum have raised controversies and contradictions over the past several years. A different and novel chemical approach to this important problem is desirable at this point in time. Recently, the molecular basis of drug resistance in P. falciparum has been associated with mutations in the resistance genes, Chloroquine Resistance Transporter (PfCRT) and the P-glycoprotein homologue (Pgh1). Although not the determinant of chloroquine resistance in P. falciparum, mutations in Pgh1 have important implications for resistance to other antimalarial drugs. Because it is mutations in the aforementioned resistance genes rather than overexpression that has been associated with drug resistance in malaria, studies on mechanisms of drug resistance and its reversal by chemosensitisers should benefit from a chemical approach. Target-oriented organic synthesis of chemosensitisers against proteins implicated in drug resistance in malaria should shed light on mechanism of drug resistance and its reversal in this area. The effect of structurally diverse chemosensitisers should be examined on several putative resistance genes in P. falciparum to deal with antimalarial drug resistance in the broadest sense. Therefore, generating random mutations of these resistance proteins and subsequent screening in search of a specific phenotype followed by a search for mutations and/or chemosensitisers that affect a specific drug resistance pathway might be a viable strategy. This diversity-oriented organic synthesis approach should offer the means to simultaneously identify resistance proteins that can serve as targets for therapeutic intervention (therapeutic target validation) and chemosensitisers that modulate the functions of these proteins (chemical target validation).     

A genetic screen identifies genes essential for development of myelinated axons in zebrafish

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.     

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.     

Plasmodium sporozoite invasion into insect and mammalian cells is directed by the same dual binding system

Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.     

Inhibition and stimulation of growth of Entamoeba histolytica in culture: association with PKC activity and protein phosphorylation

We studied the role of protein kinase C (PKC) and protein threonine phosphorylation in the inhibition and stimulation of growth of the protozoan parasite Entamoeba histolytica. PKC was activated after serum deprivation in E. histolytica and during this period proteins became threonine phosphorylated. Conversely, on serum stimulation of serum-deprived cells, PKC activation was rapidly reversed and the threonine phosphorylation of proteins quickly declined. Growth of E. histolytica was not affected by either PKC inhibitors H-7 and GF109203X or by down-regulation of PKC by Phorbol 12-Myristate 13-Acetate (PMA). Interestingly, very low doses of PMA which caused activation of PKC and were unable to down-regulate PKC after 48 h of culture, negatively influenced the growth of E. histolytica. Serine/threonine phosphatase inhibitors Okadaic acid and Calyculin A drastically inhibited growth of E. histolytica. In conclusion, the growth of E. histolytica is not adversely affected by PKC down-regulation. On the contrary, growth inhibition of E. histolytica is associated with activation of Ca(2+), Diacylglycerol (DAG)-dependent PKC, and threo nine phosphorylation of proteins.     

Entamoeba histolytica: a simplified method to quantify its cytotoxicity

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.     

Recognition of Entamoeba histolytica 115-kDa surface protein by human secretory immunoglobulin A antibodies from asymptomatic carriers

Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.     

Progress and prospects of gene inactivation in Entamoeba histolytica

Over the last few years, numerous methods have been exploited in the attempt to study Entamoeba histolytica gene functions. Yet several features of E. histolytica, like their variable DNA content and complex ploidity have made it difficult to perform classical genetic studies such as homologous recombination. As a result, the methods currently in use target genes at the protein or RNA level. This review summarizes the experimental approaches that have been used to date and it provides an overview of the limitations and contributions of these methods in our understanding of E. histolytica's gene functions and biology.     

Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach

Phagocytosis of human cells is a crucial activity for the virulence of the human parasite Entamoeba histolytica. This protozoan invades and destroys the intestine by killing and phagocytosing epithelial cells, erythrocytes and cells from the immune system. In this study, we used magnetic beads covered with proteins from human serum as a model system to study the early events involved in phagocytosis by E. histolytica. We validated the system showing that the beads uptake triggered the activation of the actin-myosin cytoskeleton and involved a PI3-kinase as previously described for erythrophagocytosis. We purified early phagosomes from wild-type (WT) amoeba and from parasites that overproduced myosin IB (MyoIB+), the unique unconventional myosin of E. histolytica. The MyoIB+ cells exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by liquid chromatography and tandem mass spectroscopy (LC-MS/MS) of the WT and MyoIB+ phagosomes allowed us to identify, for the first time, molecular actors involved in the early step of the uptake process. These include proteins involved in cytoskeleton activity, signalling, endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically recruited on the phagosomes from the MyoIB+ strain were previously described in other eukarytotic cells, as involved in the regulation of cortical F-actin dynamics, such as alpha-actinin and formins. This proteomics approach allows a step further towards the understanding of the molecular mechanisms involved in phagocytosis in E. histolytica that revealed some interesting differences compared with phagocytosis in macrophages or Dictyostelium discoideum, and allowed to identify putative candidates for proteins linked to myosin IB activity during the phagocytic process.