Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein,Cp-P34, unique to Cryptosporidium

Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.     

Proteomic characterization of sperm radial spokes identifies a novel spoke protein with an ubiquitin domain

Radial spokes are T-shaped protein complexes important for the regulation of axonemal dyneins in eukaryotic cilia and flagella. Using a functional proteomics approach, we identified six spoke proteins in sperm flagella of the ascidian Ciona intestinalis. Many of the domain/motif structures in spoke proteins are commonly found in flagella of both Ciona sperm and Chlamydomonas, but interestingly they often distribute over non-orthologous protein components. A novel 116 kDa protein named CMUB116 has both an ubiquitin domain and an IQ motif. It has orthologs in vertebrates, but not in Chlamydomonas. Furthermore, the results obtained by immunological analysis provide strong indication that CMUB116 is located at the stalk of radial spokes, where it is associated with MORN40.

Structured summary

MINT-7148244: CMUB116 (genbank_protein_gi:BAH59277) and MORN40 (genbank_protein_gi:BAH59284) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7148179: Ci-RSP3 (uniprotkb:Q8T898) physically interacts (MI:0915) with tubulin alpha (uniprotkb:Q8MVT7), LRR37 (uniprotkb:Q8T896), CMUB116 (genbank_protein_gi:BAH59277), Ci-SRP4/6 (genbank_protein_gi:BAH59283), AK58 (genbank_protein_gi:BAM59278), tubulin beta (genbank_protein_gi:XP_002130315), NDK/DPY26 (genbank_protein_gi:BAH59279), MORN40 (genbank_protein_gi:BAH59284), ARM37 (genbank_protein_gi:BAH59280), NDK/DPY26 (genbank_protein_gi:NP_001161489), LC8 (genbank_protein_gi:BAH59282) and Ci-RSP9 (genbank_protein_gi:NP_001154962) by anti bait coimmunoprecipitation (MI:0006)MINT-7148272: Ci-RSP3 (uniprotkb:Q8T898) and MORN40 (genbank_protein_gi:BAH59284) colocalize (MI:0403) by cosedimentation (MI:0027)     

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Multiple repeats of membrane occupation and recognition nexus （MORN） motifs were detected in plant phosphatidylinositl monophosphate kinase （PIPK）, a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs （with varied numbers at ranges of 7-9）, which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an -100 amino-acid ＂island＂ region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET （sequence of membrane-targeting） system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK 1, together with the 104 amino-acid ＂island＂ region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI（4,5）P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.     

Infantile-onset ascending hereditary spastic paraplegia with bulbar involvement due to the novel ALS2 mutation c.2761C > T

Recessive mutations in the alsin gene cause three clinically distinct motor neuron diseases: juvenile amyotrophic lateral sclerosis (ALS2), juvenile primary lateral sclerosis (JPLS) and infantile-onset ascending hereditary spastic paraplegia (IAHSP). A total of 23 different ALS2 mutations have been described for the three disorders so far. Most of these mutations result in a frameshift leading to a premature truncation of the alsin protein. We report the novel ALS2 truncating mutation c.2761C > T; p.R921X detected by homozygosity mapping and sequencing in two infants affected by IAHSP with bulbar involvement. The mutation c.2761C > T resides in the pleckstrin domain, a characteristic segment of guanine nucleotide exchange factors of the Rho GTPase family, which is involved in the overall neuronal development or maintenance. This study highlights the importance of using homozygosity mapping combined with candidate gene analysis to identify the underlying genetic defect as in this Saudi consanguineous family.     

Structure of the MORN4/Myo3a Tail Complex Reveals MORN Repeats as Protein Binding Modules

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Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein‐encoding genes, some of which are specifically up‐regulated during conjugation. A lipid‐binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.     

Trypanosomatid Pin1‐Type Peptidyl‐Prolyl Isomerase Is Cytosolic and Not Essential for Cell Proliferation

Pin1‐type peptidyl‐prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)‐Pro residues, regulating various cellular events. Previously, we reported a Pin1‐type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1‐like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi‐mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two‐hybrid assay, we identified a MORN domain‐containing protein as putative Pin1‐binding partners. These data suggest that Pin1‐mediated signaling mechanism plays a different role in protozoan parasites.