Identification of Pex34p as a component of the peroxisomal de novo biogenesis machinery in yeast

Cells can regulate the abundance and composition of peroxisomes to adapt to environmental changes. In the bakers yeast, S. cerevisiae, peroxisomes represent the only site for degradation of fatty acids. Hence, it is not surprising that growth of yeast cells on oleic acid results in a massive proliferation of peroxisomes. New peroxisomes can form either by division of pre-existing peroxisomes or de novo in a Pex25p-dependent process with the involvement of the Endoplasmic Reticulum (ER). In search for further factors involved in de novo formation of peroxisomes, we screened nearly 6,000 yeast mutants that were depleted of peroxisomes by conditional inhibition of PEX19 expression. Screening the mutants for the reappearance of peroxisomes upon expression of PEX19 identified Pex34p, in addition to the well-known component Pex25p, as crucial determinants for de novo biogenesis. Pex34p interacts with Pex19p and with different Peroxisomal Membrane Proteins (PMPs) in a PEX19-dependent manner. Depletion of Pex34p results in reduced numbers of import-competent peroxisomes formed de novo and Pex3p is partly retained and distributed in ER-like structures. We suggest that Pex25p and Pex34p are both required to maintain peroxisome number in a cell and that they perform non-redundant roles in the de novo formation of peroxisomes.

Kap-β2/Transportin mediates β-catenin nuclear transport in Wnt signaling

Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer highlighting its importance for disease pathogenesis. β-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of β-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that β-catenin accumulates in the nucleus in a Ran dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the orthologue of Kap- β2/Transportin-1 (TNPO1), was required for β-catenin nuclear import. We further demonstrate direct binding between TNPO1 and β-catenin that is mediated by a conserved amino acid sequence that resembles a PY NLS. Finally, using Xenopus secondary axis and TCF/LEF reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the NLS in β-catenin and its cognate NTR, our study provides new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step towards therapeutics.

Flagellum-Mediated Mechanosensing and RflP Control Motility State of Pathogenic Escherichia coli

ABSTRACT Bacterial flagellar motility plays an important role in many processes that occur at surfaces or in hydrogels, including adhesion, biofilm formation, and bacterium-host interactions. Consequently, expression of flagellar genes, as well as genes involved in biofilm formation and virulence, can be regulated by the surface contact. In a few bacterial species, flagella themselves are known to serve as mechanosensors, where an increased load on flagella experienced during surface contact or swimming in viscous media controls gene expression. In this study, we show that gene regulation by motility-dependent mechanosensing is common among pathogenic Escherichia coli strains. This regulatory mechanism requires flagellar rotation, and it enables pathogenic E. coli to repress flagellar genes at low loads in liquid culture, while activating motility in porous medium (soft agar) or upon surface contact. It also controls several other cellular functions, including metabolism and signaling. The mechanosensing response in pathogenic E. coli depends on the negative regulator of motility, RflP (YdiV), which inhibits basal expression of flagellar genes in liquid. While no conditional inhibition of flagellar gene expression in liquid and therefore no upregulation in porous medium was observed in the wild-type commensal or laboratory strains of E. coli, mechanosensitive regulation could be recovered by overexpression of RflP in the laboratory strain. We hypothesize that this conditional activation of flagellar genes in pathogenic E. coli reflects adaptation to the dual role played by flagella and motility during infection. IMPORTANCE Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.

Long Term Pharmacological Perturbation of Autophagy in Mice: Are HCQ Injections a Relevant Choice?

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whose loss-of-function has been linked to a growing list of pathologies. Knockout mouse models of key autophagy genes have been instrumental in the demonstration of the critical functions of autophagy, but they display early lethality, neurotoxicity and unwanted autophagy-independent phenotypes, limiting their applications for in vivo studies. To avoid problems encountered with autophagy-null transgenic mice, we investigated the possibility of disturbing autophagy pharmacologically in the long term. Hydroxychloroquine (HCQ) ip injections were done in juvenile and adult C57bl/6j mice, at range doses adapted from the human malaria prophylactic treatment. The impact on autophagy was assessed by western-blotting, and juvenile neurodevelopment and adult behaviours were evaluated for four months. Quite surprisingly, our results showed that HCQ treatment in conditions used in this study neither impacted autophagy in the long term in several tissues and organs nor altered neurodevelopment, adult behaviour and motor capabilities. Therefore, we recommend for future long-term in vivo studies of autophagy, to use genetic mouse models allowing conditional inhibition of selected Atg genes in appropriate lineage cells instead of HCQ treatment, until it could be successfully revisited using higher HCQ doses and/or frequencies with acceptable toxicity.

PfClpC Is An Essential Clp Chaperone Required For Plastid Integrity And Clp Protease Stability In Plasmodium falciparum

SummaryThe deadly malaria parasite, Plasmodium falciparum, contains a non-photosynthetic plastid known as the apicoplast, that functions to produce essential metabolites. Little is known about its biology or regulation, but drugs that target the apicoplast are clinically effective. Using phylogenetic analysis, we identified a putative complex of clp (caseinolytic protease) genes. We genetically targeted members of this complex and generated conditional mutants of the PfClpC chaperone and PfClpP protease and found that they co-localize in the apicoplast. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss, and was rescued by addition of the essential apicoplast-derived metabolite, IPP. Using a double conditional-mutant parasite line, we discovered that the chaperone activity is required to stabilize the active protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

RESTRAIN OF FEAR: PARTICIPATION OF GABA NEUROTRANSMITTER SYSTEM

In experiences on rats in the conditions of free behavior at development of a conditioned of passive avoidanсe reflex (the first series) and a defensive reflex and a conditional inhibition (the second series) it is revealed, and elaboration of internal inhibition and Phenibut – a nonspecific agonist of GAMKA and GAMKB receptors cause in experimental animals weakening of freezing arising in a dangerous situation, and a disinhibition of research behavior. Results of experiences in the accounting of data of the literature allow to assume that both factors, and elaboration of internal inhibition, and Phenibut weaken freezing – the phenomenon used in experiments as a biological analog of fear, owing to increase of level of activity of the GABA neurotransmitter system of a brain. Key words: training, freezing, fear, inhibition, disinhibition, GABA, Phenibut.