Biology subjects

Washburn, M. P.

Publications and source records attributed to Washburn, M. P..

7 recordsLinked to original sources

bioRxiv2018

The ULK1-FBXW5-SEC23B nexus controls autophagy

In response to nutrient deprivation, the cell needs to mobilize an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and a source for LC3 lipidation, COPII (Coat Protein Complex II) localizes to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and promotes autophagosome biogenesis. However, the molecular mechanisms that, in response to starvation, divert COPII from the secretory pathway to the autophagic pathway are largely unknown. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting its degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ERGIC, promoting autophagic flux. Induction of autophagy and localization of both SEC23B and SEC24B to the ERGIC in response to nutrient deprivation are significantly reduced in SEC23B(S186A) knock-in cells. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

cell biology↗

bioRxiv2018

RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

cancer biology↗

bioRxiv2018

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human DYRK1A gene encoding Dual-specificity tyrosine (Y)- Regulated Kinase 1A (DYRK1A) is a dosage-dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant of the DNA damaging therapy response.

molecular biology↗

bioRxiv2018

Ataxin-7 and non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

Ataxin-7 (Atxn7), a subunit of the SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the amino terminus of Atxn7 anchors the Non-stop deubiquitinase to the complex. To understand the consequences of Atxn7-dependent regulation of Non-stop, we sought substrates for Non-stop and discovered the deubiquitinase, dissociated from SAGA, interacts with Arp2/3 and WAVE regulatory complexes (WRC). Protein levels of WRC subunit suppressor of extracellular cAMP receptor (cAR) (SCAR) are regulated by a constant ubiquitination/proteasomal degradation mechanism. Loss of Atxn7 frees Non-stop from SAGA, leading to increased Non-stop interaction with SCAR and also increased SCAR protein levels. A Non-stop enzymatic pocket mutation that increases binding to ubiquitin increased interaction with SCAR, while an enzymatic pocket mutation reducing binding to ubiquitin also reduced binding to SCAR. Loss of Non-stop increased polyubiquitination of SCAR and reduced SCAR protein levels although SCAR protein levels were rescued by protease inhibition. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop overexpression increased SCAR protein levels and directed subcellular localization of SCAR, leading to decreased cell area and decreased number of protrusions. In vivo, heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR to produce F actin, but heterozygous mutation of SCAR did not significantly rescue retinal axon mistargeting upon knockdown of Atxn7.\n\nSummarySAGA subunits Ataxin-7 and Non-stop regulate stability and subcellular localization of WRC subunit SCAR. Loss of Ataxin-7 increases, while loss of Non-stop decreases, SCAR protein levels and F-actin network assembly.

cell biology↗

bioRxiv2018

A chemoproteomic portrait of the oncometabolite fumarate

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth and survival. Understanding these mechanisms and developing biomarkers to identify their presence thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of the covalent targets of fumarate, an oncometabolite whose accumulation marks the genetic cancer predisposition syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). First, we validate the ability of known and novel chemoproteomic probes to report on fumarate reactivity in vitro. Next, we apply these probes in concert with LC-MS/MS to identify cysteine residues sensitive to either fumarate treatment or fumarate hydratase (FH) mutation in untransformed and human HLRCC cell models, respectively. Mining this data to understand the structural determinants of fumarate reactivity reveals an unexpected anti-correlation with nucleophilicity, and the discovery of a novel influence of pH on fumarate-cysteine interactions. Finally, we show that many fumarate-sensitive and FH-regulated cysteines are found in functional protein domains, and perform mechanistic studies of a fumarate-sensitive cysteine in SMARCC1 that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the influence of fumarate on reactive cysteine residues, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

cancer biology↗

bioRxiv2017

The Human Cytoplasmic Dynein Interactome Reveals Novel Activators Of Motility

In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome or \"transportome\" by attaching a promiscuous biotin ligase (\"BioID\") to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires \"activators\", of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos.

cell biology↗

bioRxiv2016

A new coactivator complex required for retinoic acid-dependent regulation of embryonic symmetry

Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires Retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signalling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1 and Hdac2 are required for RA signalling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA Polymerase II recruitment. Our work identifies a novel protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.

developmental biology↗