Asparagine Repeats Research Articles

Chaperoning of asparagine repeat-containing proteins in Plasmodium falciparum.

Plasmodium falciparum has the most adenine (A)- and thymine (T)-rich genome known to date, and 24-30% of the P. falciparum proteome contains asparagine (N) and glutamine (Q) residues. In general, asparagine repeats in proteins increase the propensity for aggregation, especially at elevated temperatures, which occur routinely in P. falciparum parasites during exoerythrocytic and erythrocytic developmental stages in a vertebrate host. The P. falciparum exported chaperone machinery is comprised of an exported PfHsp70-x protein and its co-chaperone PfHsp40-x1 in the host erythrocyte compartment, and PfHsp70-z and its co-chaperones in the parasite cytoplasm have been identified. In vitro assays reveal that these chaperone partners function in refolding of asparagine-rich polypeptides. The identification and chaperoning of exported poly-asparagine-containing proteins, and the biological roles and the protection mechanisms of P. falciparum during febrile conditions by the exported chaperone machinery are discussed.

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum.

Asparagine Repeat Peptides: Aggregation Kinetics and Comparison with Glutamine Repeats.

Amino acid repeat runs are common occurrences in eukaryotic proteins, with glutamine (Q) and asparagine (N) as particularly frequent repeats. Abnormal expansion of Q-repeat domains causes at least nine neurodegenerative disorders, most likely because expansion leads to protein misfolding, aggregation, and toxicity. The linkage between Q-repeats and disease has motivated several investigations into the mechanism of aggregation and the role of Q-repeat length in aggregation. Curiously, glutamine repeats are common in vertebrates, whereas N-repeats are virtually absent in vertebrates, but common in invertebrates. One hypothesis for the lack of N-repeats in vertebrates is biophysical; that is, there is strong selective pressure in higher organisms against aggregation-prone proteins. If true, then asparagine and glutamine repeats must differ substantially in their aggregation properties despite their chemical similarities. In this work, aggregation of peptides with asparagine repeats of variable length (12-24) were characterized and compared to that of similar peptides with glutamine repeats. As with glutamine, aggregation of N-repeat peptides was strongly length-dependent. Replacement of glutamine with asparagine caused a subtle shift in the conformation of the monomer, which strongly affected the rate of aggregation. Specifically, N-repeat peptides adopted β-turn structural elements, leading to faster self-assembly into globular oligomers and much more rapid conversion into fibrillar aggregates, compared to Q-repeat peptides. These biophysical differences may account for the differing biological roles of N- versus Q-repeat domains.

Synthesis and disaggregation of asparagine repeat‐containing peptides

Of all amino acid repeats in eukaryotes, polyglutamine (polyQ) is the most frequent, followed by polyasparagine (polyN). Glutamine repeats are expanded in proteins associated with several neurodegenerative disorders. The expanded polyQ domain is known to induce aggregation, and it is hypothesized that aggregation is directly causative of pathology. Despite the widespread presence of asparagine repeats in invertebrate eukaryotes, polyN is curiously quite rare in vertebrates. Several investigators have characterized the conformational and aggregation properties of polyQ-containing peptides and proteins, and to a lesser extent, peptides containing mixed glutamine and asparagine, but to our knowledge, there is no detailed characterization of polyN-containing peptides. Such a comparison could elucidate reasons for the paucity of asparagine repeats in humans. In this study, we synthesized a peptide containing a 24-asparagine repeat (N24). For aggregation studies, it is critical to start with monomeric unaggregated peptide. A protocol involving dissolution in mixed trifluoroacetic acid and hexafluoroisopropanol (TFA + HFIP) solvents is widely used for disaggregation of polyQ peptides. We used the same protocol for N24 but discovered that there was both oxidative damage and insufficient disaggregation. Oxidation of tryptophan, used as a flanking residue, was common. Moreover, we found evidence of Förster resonance energy transfer between Trp and its oxidation product N-formylkynurenine, even in chemical denaturants. This suggested that N24 was insufficiently disaggregated, a conclusion that was further supported by gel electrophoresis analysis. Oxidation was reduced, but not eliminated, by addition of methionine to the buffer. Formic acid proved to be a better disaggregator and caused no oxidative damage. The glutamine repeat peptide Q24 also underwent some oxidation after extended incubation in TFA + HFIP, but there was no evidence of Förster resonance energy transfer, and samples appeared monomeric by gel electrophoresis. This result indicates that polyN-containing peptides self-associate more strongly than polyQ-containing peptides. Circular dichroism spectra reveal a greater propensity for β-turn formation in polyN than polyQ, providing an explanation for the increased stability of polyN aggregates relative to polyQ.

Aminoacylation of Plasmodium falciparum tRNAAsn and Insights in the Synthesis of Asparagine Repeats

Genome sequencing revealed an extreme AT-rich genome and a profusion of asparagine repeats associated with low complexity regions (LCRs) in proteins of the malarial parasite Plasmodium falciparum. Despite their abundance, the function of these LCRs remains unclear. Because they occur in almost all families of plasmodial proteins, the occurrence of LCRs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. Translation of these asparagine-rich LCRs demands extraordinarily high amounts of asparaginylated tRNA(Asn). However, unlike other organisms, Plasmodium codon bias is not correlated to tRNA gene copy number. Here, we studied tRNA(Asn) accumulation as well as the catalytic capacities of the asparaginyl-tRNA synthetase of the parasite in vitro. We observed that asparaginylation in this parasite can be considered standard, which is expected to limit the availability of asparaginylated tRNA(Asn) in the cell and, in turn, slow down the ribosomal translation rate when decoding asparagine repeats. This observation strengthens our earlier hypothesis considering that asparagine rich sequences act as "tRNA sponges" and help cotranslational folding of parasite proteins. However, it also raises many questions about the mechanistic aspects of the synthesis of asparagine repeats and about their implications in the global control of protein expression throughout Plasmodium life cycle.

Perturbation Of Gpsm2/Lgn Enhances Haematopoietic Stem Cell Function

A gain of function screen of candidate cell polarity genes identified the gene, Gpsm2 (G protein signaling modulator 2) also known as LGN or Pins, as an enhancer of haematopoietic stem cell (HSC) function. Mammalian Gpsm2 comprises an N-terminal domain composed of ten Leucine–Glycine–Asparagine (LGN) repeats within seven to eight tetratricopeptide motifs, which functions to bind NuMA (Nuclear Mitotic Apparatus) and a C-terminal domain containing four GoLoco repeats that modulates guanine nucleotide exchange via Gai interaction. In effect, Gpsm2 is an integral component that coordinates G protein signalling to mitotic spindle orientation during cell division. In multiple independent experiments, Gpsm2-transduced HSCs and cultured for 7 days in vitro compared to vector-transduced HSCs showed enhanced HSC repopulation out to 20 weeks-plus post transplant. At these time points, bone marrow and thymic DNA from multiple recipient mice were analysed via Southern blots with a GFP probe. This showed Gpsm2-transduced HSCs retained multipotency but that only one out of a minimum of 12 clones had undergone in vitro symmetrical expansion. Current limiting dilution assay will assess quantitatively this relative absence of HSC expansion. Although in vitro assays with Gpsm2-transduced bone marrow cells showed reduced colony formation and slightly attenuated cell proliferation, limiting dilution assays in secondary transplants showed Gpsm2-transduced HSCs maintained self-renewal ability together with a significant (p=0.009) 4-fold increase in HSC frequency compared to vector-transduced HSCs. This presumably reflects the importance of the interaction between Gpsm2-transduced HSCs within the niche in vivo. A mouse mutant lacking all GoLoco repeats of the C-terminal domain (LGN deltaC) is viable, where analyses of the neuroprogenitors of this mouse revealed altered asymmetrical versus symmetrical cell divisions without a detrimental effect on neuronal production (Konno et al. Nat Cell Biol 2008). A subsequent study of the role of LGN in epidermal development identified that a significant (>80%) knockdown of LGN via shRNA also altered asymmetrical cell divisions of epidermal stem-progenitor cells, however with a functional loss of skin barrier (Williams et al. Nature 2011). This defect could be partially rescued by the LGN N-terminal protein suggesting the LGN deltaC mouse still has functional, albeit, incomplete LGN/Gpsm2 function. We have analysed haematopoiesis in the LGN deltaC mouse. There were no significant differences in wild-type versus homozygous LGN deltaC LSK numbers, in vitro colony formation or in vivo CFU-spleen assays. However, limiting dilution assays showed a 7-fold increase (p=0.02) in HSC frequency in the LGN deltaC mouse, and competitive CD45.2/CD45.1 transplantations analysed at 16 weeks post transplant showed CD45.2 LGN deltaC HSCs reconstituted significantly (p=0.015) better compared to CD45.2 wild-type HSCs. On the premise that the Gpsm2 function of controlling somatic stem cell fate is conserved, we are investigating the hypothesise that the mechanism of perturbation of Gpsm2 enhancement of HSC function is through altered HSC asymmetric and symmetric divisional fate. Disclosures:No relevant conflicts of interest to declare.

Repeat polymorphisms in the low-complexity regions of Plasmodium falciparum ABC transporters and associations with in vitro antimalarial responses.

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.

Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers.

One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.

Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.