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1.
bioRxiv ; 2023 Jun 11.
Article in English | MEDLINE | ID: mdl-37333418

ABSTRACT

During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. We hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. We combined cell type-specific transcriptomics with a genetic screen to discover such factors. We identified Telomeric Zinc finger-Associated Protein (TZAP) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1 . In Drosophila , loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. Our findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.

2.
Sci Adv ; 9(24): eadd5002, 2023 06 16.
Article in English | MEDLINE | ID: mdl-37327344

ABSTRACT

Neurogenesis 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 species-specific 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.


Subject(s)
Amyloid beta-Protein Precursor , Neural Stem Cells , Humans , Mice , Animals , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Cell Differentiation , Neurons/metabolism , Neurogenesis
3.
Cell Rep ; 35(10): 109208, 2021 06 08.
Article in English | MEDLINE | ID: mdl-34107249

ABSTRACT

Brain 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.


Subject(s)
Cerebellum/physiology , Neurons/metabolism , Receptors, Notch/metabolism , Cell Differentiation , Humans
4.
J Mol Biol ; 430(11): 1545-1558, 2018 05 25.
Article in English | MEDLINE | ID: mdl-29694833

ABSTRACT

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.


Subject(s)
Membrane Proteins/metabolism , Peroxisomes/metabolism , Pichia/metabolism , Fungal Proteins/metabolism , Peroxins/genetics , Peroxins/metabolism , Peroxisomes/genetics , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
5.
Biochem Biophys Res Commun ; 496(2): 562-567, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29288668

ABSTRACT

Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.


Subject(s)
Fungal Proteins/metabolism , Peroxins/metabolism , Pichia/metabolism , Catalytic Domain , Crystallography, X-Ray , Fungal Proteins/chemistry , Models, Molecular , Peroxins/chemistry , Pichia/chemistry , Protein Binding , Protein Conformation , Ubiquitination , Ubiquitins/metabolism
6.
Chem Commun (Camb) ; 53(87): 11929-11932, 2017 Oct 31.
Article in English | MEDLINE | ID: mdl-29046906

ABSTRACT

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.

7.
Methods Mol Biol ; 1595: 233-241, 2017.
Article in English | MEDLINE | ID: mdl-28409467

ABSTRACT

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.


Subject(s)
Membrane Proteins/metabolism , Peroxisomes/metabolism , Ubiquitinated Proteins/metabolism , Yeasts/metabolism , Blotting, Western , Immunoprecipitation , Protein Transport , Ubiquitin/metabolism , Ubiquitination
8.
ACS Nano ; 11(5): 4387-4394, 2017 05 23.
Article in English | MEDLINE | ID: mdl-28353339

ABSTRACT

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.


Subject(s)
Molecular Imaging/methods , Nanotechnology/methods , Biological Products , Nanopores , Perforin/metabolism , Protein Processing, Post-Translational , Proteins/metabolism , Ubiquitin , Ubiquitination/physiology
9.
Cancer Res ; 72(24): 6414-24, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23087057

ABSTRACT

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 G(1) to 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 PARP. We found 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.


Subject(s)
Biomarkers, Pharmacological , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Neoplasms/diagnosis , Poly(ADP-ribose) Polymerase Inhibitors , Protein Phosphatase 2/genetics , Recombinational DNA Repair/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Pharmacological/metabolism , Biomarkers, Tumor/genetics , Biomarkers, Tumor/physiology , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Down-Regulation/genetics , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/therapeutic use , Gene Deletion , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Prognosis , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/physiology , Treatment Outcome
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