Two Plasmodium Rhomboid Proteases Preferentially Cleave Different Adhesins Implicated in All Invasive Stages of Malaria

Invasion of host cells by the malaria pathogen Plasmodium relies on parasite transmembrane adhesins that engage host-cell receptors. Adhesins must be released by cleavage before the parasite can enter the cell, but the processing enzymes have remained elusive. Recent work indicates that the Toxoplasma rhomboid intramembrane protease TgROM5 catalyzes this essential cleavage. However, Plasmodium does not encode a direct TgROM5 homolog. We examined processing of the 14 Plasmodium falciparum adhesins currently thought to be involved in invasion by both model and Plasmodium rhomboid proteases in a heterologous assay. While most adhesins contain aromatic transmembrane residues and could not be cleaved by nonparasite rhomboid proteins, including Drosophila Rhomboid-1, Plasmodium falciparum rhomboid protein (PfROM)4 (PFE0340c) was able to process these adhesins efficiently and displayed novel substrate specificity. Conversely, PfROM1 (PF11_0150) shared specificity with rhomboid proteases from other organisms and was the only PfROM able to cleave apical membrane antigen 1 (AMA1). PfROM 1 and/or 4 was thus able to cleave diverse adhesins including TRAP, CTRP, MTRAP, PFF0800c, EBA-175, BAEBL, JESEBL, MAEBL, AMA1, Rh1, Rh2a, Rh2b, and Rh4, but not PTRAMP, and cleavage relied on the adhesin transmembrane domains. Swapping transmembrane regions between BAEBL and AMA1 switched the relative preferences of PfROMs 1 and 4 for these two substrates. Our analysis indicates that PfROMs 1 and 4 function with different substrate specificities that together constitute the specificity of TgROM5 to cleave diverse adhesins. This is the first enzymatic analysis of Plasmodium rhomboid proteases and suggests an involvement of PfROMs in all invasive stages of the malaria lifecycle, in both the vertebrate host and the mosquito vector

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

A family of Rhomboiproteases activates all Drosod intramembrane phila membrane-tethered EGF ligands

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. We have analysed four of these: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signalling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. Our data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands

An Arabidopsis Rhomboid homolog is an intramembrane protease in plants

AbstractRegulated intramembrane proteolysis (RIP) is a fundamental mechanism for controlling a wide range of cellular functions. The Drosophila protein Rhomboid-1 (Rho-1) is an intramembrane serine protease that cleaves epidermal growth factor receptor (EGFR) ligands to release active growth factors. Despite differences in the primary structure of Rhomboid proteins, the proteolytic activity and substrate specificity of these enzymes has been conserved in diverse organisms. Here, we show that an Arabidopsis Rhomboid protein AtRBL2 has proteolytic activity and substrate specificity. AtRBL2 cleaved the Drosophila ligands Spitz and Keren, but not similar proteins like TGFα, when expressed in mammalian cells, leading to the release of soluble ligands into the medium. These studies provide the first evidence that the determinants of RIP are present in plants

Specificity of PfROMs 1 and 4 for the Transmembrane Domains of AMA1 versus that of BAEBL

<div><p>(A) Cleavage of GFP-tagged BAEBL (drawn in black) and GFP-tagged BAEBL harboring the transmembrane domain of AMA1 (in red) were tested for cleavage by anti-GFP Western analysis of media from transiently transfected COS cells. While TgROM5 could cleave both proteins efficiently, PfROM1 could only cleave BAEBL + AMAtm, while PfROM4 could only cleave BAEBL.</p><p>(B) Cleavage of GFP-tagged AMA1 (drawn in red) and GFP-tagged AMA1 harboring the transmembrane domain of BAEBL (in black) were tested for cleavage by anti-GFP Western analysis of media from transiently transfected COS cells. While TgROM5 could cleave both proteins efficiently, PfROM1 could only cleave AMA1 while PfROM4 could only cleave AMA + BAEBLtm.</p><p>(C) Other rhomboid enzymes including YqgP (from Bacillus subtilis) and RHBDL2 (from Homo sapiens) also cleaved BAEBL more efficiently when it contained the transmembrane domain from AMA1, like PfROM1 but not PfROM4. Molecular weight standards in kDa are denoted to the right of each panel.</p></div

Cleavage of Rh Proteins by Parasite Rhomboid Enzymes

<p>GFP-tagged Rh1 (A), Rh2a (B), Rh2b (C), and Rh4 (D) were tested for cleavage by rhomboid enzymes by analyzing conditioned media with anti-GFP from transiently transfected COS cells. TgROM5 and PfROM4 readily cleaved each Rh protein, while PfROM1 could not cleave Rh2a but cleaved other Rh proteins at a lower efficiency. Note that in many cases smaller GFP-Rh protein breakdown products were readily detected in conditioned media (unpublished data). Molecular weight standards in kDa are denoted to the right of each panel.</p

Cleavage of MAEBL and CTRP Adhesins, Which Are Required for Invasion of Mosquito Cells, by Parasite Rhomboid Enzymes

<p>GFP-tagged MAEBL (A) and CTRP (B) were cotransfected with each rhomboid, and conditioned media were analyzed by anti-GFP Western. Molecular weight standards in kDa are denoted to the right of each panel.</p