Insights into the Role of the Peroxisomal Ubiquitination Machinery in Pex13p Degradation in the Yeast Hansenula polymorpha

The import of matrix proteins into peroxisomes in yeast requires the action of the ubiquitin-conjugating enzyme Pex4p and a complex consisting of the ubiquitin E3 ligases Pex2p, Pex10p and Pex12p. Together, this peroxisomal ubiquitination machinery is thought to ubiquitinate the cycling receptor protein Pex5p and members of the Pex20p family of co-receptors, a modification that is required for receptor recycling. However, recent reports have demonstrated that this machinery plays a role in additional peroxisome-associated processes. Hence, our understanding of the function of these proteins in peroxisome biology is still incomplete. Here, we identify a role for the peroxisomal ubiquitination machinery in the degradation of the peroxisomal membrane protein Pex13p. Our data demonstrate that Pex13p levels build up in cells lacking members of this machinery and also establish that Pex13p undergoes rapid degradation in wild-type cells. Furthermore, we show that Pex13p is ubiquitinated in wild-type cells and also establish that Pex13p ubiquitination is reduced in cells lacking a functional peroxisomal E3 ligase complex. Finally, deletion of PEX2 causes Pex13p to build up at the peroxisomal membrane. Taken together, our data provide further evidence that the role of the peroxisomal ubiquitination machinery in peroxisome biology goes much deeper than receptor recycling alone

Detection of Ubiquitinated Peroxisomal Proteins in Yeast

Ubiquitination is involved in different aspects of peroxisome formation, maintenance, and degradation. Consequently, simple methods for detecting ubiquitinated peroxisomal proteins are extremely useful in peroxisomal research. Here, we describe an immunoprecipitation-based technique that can be used to assess peroxisomal protein ubiquitination in yeast

Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination

Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

The covalent addition of ubiquitin to target proteins is a key post-translational modification that is linked to a myriad of biological processes. Here, we report a fast, single-molecule, and label-free method to probe the ubiquitination of proteins employing an engineered Cytolysin A (ClyA) nanopore. We show that ionic currents can be used to recognize mono- and polyubiquitinated forms of native proteins under physiological conditions. Using defined conjugates, we also show that isomeric monoubiquitinated proteins can be discriminated. The nanopore approach allows following the ubiquitination reaction in real time, which will accelerate the understanding of fundamental mechanisms linked to protein ubiquitination.status: publishe

Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition

Reversible phosphorylation plays a critical role in DNA repair. Here we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1/S phase cell cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small molecule inhibitors of poly(ADP-ribose) polymerase (PARP). We found that that PPP2R2A was commonly downregulated in non-small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic.status: publishe

Target and identify:triazene linker helps identify azidation sites of labelled proteins via click and cleave strategy

A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA

Direct and indirect interactions between owls, mice and nocturnal seabirds: integrating marine and terrestrial food webs

Climate variability in semi-arid ecosystems can influence species interactions from the bottom-up, and through these perturbations we can gain insight into both direct and indirect interactions in food webs. In this thesis, I studied the effects of ENSO-driven rainfall pulses and drought on the interactions between a top predator, the Barn Owl (Tyto alba), a mesopredator, an island endemic deer mouse (Peromyscus maniculatus elusus), and a threatened nocturnal seabird, the Scripps’s Murrelet (Synthliboramphus scrippsi). On Santa Barbara Island in the Channel Islands National Park in California, adult breeding murrelets are killed by owls, but their eggs are eaten by mice, which is the main cause of reduced murrelet nest success. First, I assessed how owl predation on murrelets varies with the availability of mice, the primary prey of owls. I found that heavy rainfall years drive the irruptions in the mouse population that precede peaks in owl abundance, which results in high murrelet predation by owls when the mouse population subsequently crashes. Next, I examined evidence for positive indirect effects of owls on murrelets through their influence on mouse foraging behavior. I found that mouse foraging was strongly suppressed as the abundance of owls increased, and survival of murrelet eggs was also positively related to owl abundance. I also examined how both the terrestrial and marine environments influenced overall murrelet nest success over a span of 21 years. I found that the severity of drought was the most important variable determining nest success, which suggests that during severe droughts, mice consume substantially more eggs when there are fewer terrestrial resources and also less risk from predation. Climate-driven indirect interactions with predators therefore influences both survival and nest success of murrelets on this island. Finally, I developed a mathematical model of island community dynamics to assess whether owl management might benefit murrelets given projected changes to rainfall patterns in this region. I found no evidence that managing the owl population would enhance murrelet abundance, demonstrating the importance of considering both direct and indirect effects of predators when evaluating potential conservation strategies

Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum

International audienceBrain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors

The temporal balance between self-renewal and differentiation of human neural stem cells requires the amyloid precursor protein

International audienceNeurogenesis in the developing human cerebral cortex occurs at a particularly slow rate owing in part to cortical neural progenitors preserving their progenitor state for a relatively long time, while generating neurons. How this balance between the progenitor and neurogenic state is regulated, and whether it contributes to speciesspecific brain temporal patterning, is poorly understood. Here, we show that the characteristic potential of human neural progenitor cells (NPCs) to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the amyloid precursor protein (APP). In contrast, APP is dispensable in mouse NPCs, which undergo neurogenesis at a much faster rate. Mechanistically, APP cell-autonomously contributes to protracted neurogenesis through suppression of the proneurogenic activator protein-1 transcription factor and facilitation of canonical WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis