Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

A localization screen reveals translation factories and widespread co-translational RNA targeting

SummaryLocal translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

βVPE is involved in tapetal degradation and pollen development by activating proprotease maturation in Arabidopsis thaliana

Vacuolar processing enzyme (VPE) is responsible for the maturation and activation of vacuolar proteins in plants. We found that βVPE was involved in tapetal degradation and pollen development by transforming proproteases into mature protease in Arabidopsis thaliana. βVPE was expressed specifically in the tapetum from stages 5 to 8 of anther development. The βVPE protein first appeared as a proenzyme and was transformed into the mature enzyme before stages 7–8. The recombinant βVPE protein self-cleaved and transformed into a 27 kDa mature protein at pH 5.2. The mature βVPE protein could induce the maturation of CEP1 in vitro. βvpe mutants exhibited delayed vacuolar degradation and decreased pollen fertility. The maturation of CEP1, RD19A, and RD19C was seriously inhibited in βvpe mutants. Our results indicate that βVPE is a crucial processing enzyme that directly participates in the maturation of cysteine proteases before vacuolar degradation, and is indirectly involved in pollen development and tapetal cell degradation.

Neonatal Intracranial Hemorrhage with a Dramatic Outcome Due to Maternal Anti CD36 Antibodies

Fetal/neonatal allo-immune thrombocytopenia (FNAIT) results from maternal immunization against fetal platelet-specific antigens (HPA) inherited from the father. Most cases involve HPA located on glycoproteins (GP) IbIX, IaIIa and IIbIIIa. Iso-immunizations can also occur in the absence of expression of membrane proteins, such as GPIIb or GPIIIa in Glanzmann patients. CD36 (also called glycoprotein GPIV) deficiency is observed in 3 to 5% of Asian and African populations. We report here the case of a 41-year-old Canadian woman originated from Africa, who delivered a male dead new-born at 39 weeks of gestation. A massive intracranial haemorrhage was identified as being the obvious cause of death. No platelet antibody against GPIbIX, IaIIa, and IIbIIIa was identified by the gold-standard Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assay. Surprisingly, anti CD36 iso-antibodies were identified in the maternal serum with a new bead-based multiplex assay. The CD36 gene was sequenced for both parents, and a mutation was identified on Exon 10 of the mother’s CD36 gene, which was absent for the father: NM_000072.3:c.975T>G inducing a STOP codon at position 325 of the mature protein. The absence of CD36 expression on the mother’s platelets was confirmed by flow cytometry.

A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state

ABSTRACTAspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) fromTalaromyces leycettanuswas identified and successfully expressed inPichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions.TlAPA1 shared the highest identity of 70.3 % with the aspartic endopeptidase fromByssochlamys spectabilis(GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. MatureTlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature ofTlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (TlAPA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic proteaseTlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation.IMPORTANCEThe novel aspartic proteaseTlAPA1 was identified fromT. leycettanusand expressed as a zymogen (proTlAPA1) inP. pastoris. Enzymatic characteristics of the mature protein were studied and the specific pattern of zymogen conversion was described. The auto-activation processing of proTlAPA1 proceeded in two stages and an intermediate was identified in this process. These results describe a new subgroup of aspartic protease A1 family and provide insights into a novel mode of activation processing in aspartic proteases.

βVPE is involved in tapetal degradation and pollen development by activating proprotease maturation in Arabidopsis thaliana

Vacuolar processing enzyme (VPE) is responsible for the maturation and activation of vacuolar proteins in plants. We found that βVPE was involved in tapetal degradation and pollen development by transforming proproteases into mature protease in Arabidopsis thaliana. βVPE was expressed specifically in the tapetum from stages 5–8 of anther development. The βVPE protein first appeared as a proenzyme and transformed into the mature enzyme before stages 7–8. The recombinant βVPE protein self-cleaved and transformed to a 27-kD mature protein at pH 5.2. The mature βVPE protein could induce the maturation of CEP1 in vitro. βvpe mutants exhibited delayed vacuolar degradation and decreased pollen fertility. The maturation of CEP1, RD19A, and RD19C were seriously inhibited in βvpe mutants. Our results indicate that βVPE is a crucial processing enzyme that directly participates in the maturation of cysteine proteases before vacuolar degradation, and is indirectly involved in pollen development and tapetal cell degradation.

IP6 Regulation of HIV Capsid Assembly, Stability, and Uncoating

The mechanisms that drive formation of the HIV capsid, first as an immature particle and then as a mature protein shell, remain incompletely understood. Recent discoveries of positively-charged rings in the immature and mature protein hexamer subunits that comprise them and their binding to the cellular metabolite inositol hexakisphosphate (IP6) have stimulated exciting new hypotheses. In this paper, we discuss how data from multiple structural and biochemical approaches are revealing potential roles for IP6 in the HIV-1 replication cycle from assembly to uncoating.

Uroguanylin: a new actor in the energy balance movie

Uroguanylin (UGN) is a potential target in the fight against obesity. The mature protein is released after enzymatic cleavage from its natural precursor, proUGN. UGN is mostly produced in the gut, and its production is regulated by nutritional status. However, UGN is also produced in other tissues such as the kidneys. In the past, UGN has been widely studied as a natriuretic peptide owing to its involvement in several different pathologies such as heart failure, cancer and gastrointestinal diseases. However, recent studies have suggested that UGN also acts as a regulator of body weight homeostasis because it modulates both food intake and energy expenditure. This ultimately results in a decrease in body weight. This action is mediated by the sympathetic nervous system. Future studies should be directed at the potential effects of UGN agonists in regulating body weight in human obesity.