Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.     

Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.     

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4^Cre and Mrf4^nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.     

Disruption of mitotic arrest precedes precocious differentiation and transdifferentiation of pregranulosa cells in the perinatal Wnt4 mutant ovary

Mammalian sex determination is controlled by antagonistic pathways that are initially co-expressed in the bipotential gonad and subsequently become male- or female-specific. In XY gonads, testis development is initiated by upregulation of Sox9 by SRY in pre-Sertoli cells. Disruption of either gene leads to complete male-to-female sex reversal. Ovarian development is dependent on canonical Wnt signaling through Wnt4, Rspo1 and β-catenin. However, only a partial female-to-male sex reversal results from disruption of these ovary-promoting genes. In Wnt4 and Rspo1 mutants, there is evidence of pregranulosa cell-to-Sertoli cell transdifferentiation near birth, following a severe decline in germ cells. It is currently unclear why primary sex reversal does not occur at the sex-determining stage, but instead occurs near birth in these mutants. Here we show that Wnt4-null and Rspo1-null pregranulosa cells transition through a differentiated granulosa cell state prior to transdifferentiating towards a Sertoli cell fate. This transition is preceded by a wave of germ cell death that is closely associated with the disruption of pregranulosa cell quiescence. Our results suggest that maintenance of mitotic arrest in pregranulosa cells may preclude upregulation of Sox9 in cases where female sex-determining genes are disrupted. This may explain the lack of complete sex reversal in such mutants at the sex-determining stage.     

A survey of species assigned to the fossil pollen genus Anacolosidites

Anacolosidites Cookson & Pike, a fossil pollen genus recorded since the Campanian, is peculiar in its morphology – six‐porate with three apertures on each hemisphere, located away from the equator, and with the distal and proximal apertures positioned over each other. Representatives of this fossil genus are widely considered to represent extant Olacaceae from tribe Anacoloseae. Olacaceae is an exclusively tropical angiosperm family with a pantropical distribution; consequently the fossils are often used to suggest a tropical climate and in addition are frequently used as a stratigraphic marker. Fossil species assigned to Anacolosidites are quite variable and may not all represent Olacaceae, in which case they may not indicate tropical climate.

The present study is a morphological survey of fossil pollen assigned to Anacolosidites; it identifies the published reports of the fossil species that probably represent positive occurrences of Olacaceae pollen in the fossil record. Within Olacaceae, Anacolosidites‐type pollen is usually compared with pollen of genera in tribe Anacoloseae, in particular: Cathedra, Anacolosa and Phanerodiscus, but never with the pollen of Ptychopetalum, a genus from tribe Olaceae with closely similar pollen to the other three genera, but with a reticulate tectum and very small circular apertures located near the equator. Nevertheless, the records of reticulate Anacolosidites species which have been excluded from the emended diagnosis are unlikely to be related to Ptychopetalum. The earliest accepted record of Anacolosidites is from the Late Cretaceous (Maastrichtian) of Germany. However, most Late Cretaceous records, and later Russian and Chinese occurrences referred to Anacolosidites, consist mainly of Normapolles‐type pollen, whereas many of the Cenozoic records assigned to Anacolosidites have a much clearer affinity with the pollen of Anacolosa, Cathedra and Phanerodiscus (tribe Anacoloseae). The newly emended genus Anacolosidites may be used as a stratigraphic marker for tropical or megathermal climatic conditions.     

Vac14 Protein Multimerization Is a Prerequisite Step for Fab1 Protein Complex Assembly and Function

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P₂ misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P₂ whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P₂ by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P₂. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P₂ levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P₂ levels.     

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.