Plasmodium falciparum†SERA5 plays a non-enzymatic role in the malarial asexual blood-stage lifecycle.

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes.

High-throughput generation of selected reaction-monitoring assays for proteins and proteomes.

Selected reaction monitoring (SRM) uses sensitive and specific mass spectrometric assays to measure target analytes across multiple samples, but it has not been broadly applied in proteomics owing to the tedious assay development process for each protein. We describe a method based on crude synthetic peptide libraries for the high-throughput development of SRM assays. We illustrate the power of the approach by generating and applying validated SRM assays for all Saccharomyces cerevisiae kinases and phosphatases.

Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics.

Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino-acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95-99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes.

Neurobin/TMPRSS11c, a novel type II transmembrane serine protease that cleaves fibroblast growth factor-2 in vitro.

TTSPs [type II TMPRSSs (transmembrane serine proteases)] are a growing family of trypsin-like enzymes with, in some cases, restricted tissue distribution. To investigate the expression of TTSPs in the nervous system, we performed a PCR-based screening approach with P10 (postnatal day 10) mouse spinal cord mRNA. We detected the expression of five known TTSPs and identified a novel TTSP, which we designated neurobin. Neurobin consists of 431 amino acids. In the extracellular part, neurobin contains a single SEA (sea-urchin sperm protein, enterokinase and agrin) domain and a C-terminal serine protease domain. RT-PCR (reverse transcription-PCR) analysis indicated the expression of neurobin in spinal cord and cerebellum. Histochemical analysis of brain sections revealed distinct staining of Purkinje neurons of the cerebellum. Transiently overexpressed neurobin was autocatalytically processed and inserted into the plasma membrane. Autocatalytic activation could be suppressed by mutating Ser(381) in the catalytic pocket to an alanine residue. The protease domain of neurobin, produced in Escherichia coli and refolded from inclusion bodies, cleaved chromogenic peptides with an arginine residue in position P(1). Serine protease inhibitors effectively suppressed the proteolytic activity of recombinant neurobin. Ca2+ or Na+ ions did not significantly modulate the catalytic activity of the protease. Recombinant neurobin processed 17-kDa FGF-2 (fibroblast growth factor-2) at several P(1) lysine and arginine positions to distinct fragments, in a heparin-inhibitable manner, but did not cleave FGF-7, laminin or fibronectin. These results indicate that neurobin is an authentic TTSP with trypsin-like activity and is able to process FGF-2 in vitro.