Podcasts about ubiquitination

Researchers observe what ubiquitination hinges on Hello and welcome to the NanoLSI podcast. Thank you for joining us today. In this episode we feature the latest research by Hiroki Konno and Holger Flechsig at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University.The research described in this podcast was published in Nano Letters in December 2023Kanazawa University NanoLSI websitehttps://nanolsi.kanazawa-u.ac.jp/en/Researchers observe what ubiquitination hinges onResearchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University report in Nano Letters how the flexibility of a protein hinge plays a crucial role in the transfer of proteins in key cell processes.Ubiquitination – the addition of the protein ubiquitin – is a key stage in many cell processes, such as protein degradation, DNA repairs, and signal transduction. Using high-speed atomic force microscopy (AFM) and molecular modelling, researchers led by Hiroki Konno and Holger Flechsig at WPI-NanoLSI, Kanazawa University have identified how the mobility of a ubiquitination related enzyme hinge allows ubiquitination to take place.So what was known already about ubiquitination?Previous studies have identified a number of enzymes that facilitate ubiquitination, including an enzyme that activates ubiquitin (E1), an enzyme that conjugates it (E2), and an enzyme that catalyzes ubiquitin protein joining (that is, a ligase, E3) to a target protein. The HECT-type E3 ligase is characterized by a HECT domain that comprises an N lobe with the E2-binding site and a C lobe with a catalytic Cys residue, A flexible hinge connects the two lobes, leading to the hypothesis that ubiquitination is facilitated by the rearrangement of the protein around this hinge. Konno and their collaborators deployed their high-speed atomic force microscope to hunt for evidence that this was the case.So what did they find out?The researchers noted that when the HECT domain was crystallized with a type of E2 enzyme, it formed an L shape such that the distance between the catalytic Cys residue of the HECT domain and the catalytic Cys of the E2 enzyme was 41 Å – too far for the transfer of ubiquitin. However, in its catalytic conformation the HECT domain has a different shape where the distance between the two catalytic Cys residues is much closer – just 8 Å – so this is thought to be a “catalytic conformation”.Analysis of high-speed-AFM images of a wild-type HECT domain of E6AP revealed two conformations – one of which looked spherical and the other oval. Using AFM simulations they attributed the oval shapes to the L conformation and spherical shapes are either the catalytic conformation or the so called inverted T conformation, which had been observed in the another type of HECT domain where the distance between the Cys residues is 16 Å. To overcome the spatio-temporal resolution limitations of imaging, the experiments were complemented by molecular modelling to visualize HECT domain conformational motions at the atomistic level. Simulation AFM was used to generate a corresponding pseudo AFM movie, which clearly showed the change from spherical to the oval shaped topography.“Although experimental limitations do not allow us to resolve the intermediate conformations,” explain the researchers in their report of the work. “The performed modeling provides evidence that the transitions between spherical and oval HECT domain shapes observed under high-speed-AFM correspond to functional conformational motions under which the C-lobe rotates relative to the N-lobe, thereby allowing the change between catalytic and L-shape HECT conformations.”Further experiments with mutant HECT domains with less flexibility in the hNanoLSI Podcast website

While DNA captures most of the fanfare, proteins are the catalytic and structural superstars of the cell. However, they can also become problematic. Cells have intricate mechanisms to remove damaged or mis-expressed proteins that could be deleterious to cellular function. This process is mediated by a process called ubiquitination, mediated by a special class of proteins called E3 ligases. Ubiquitin is the tag that's added that signals that a protein should be moved to the biochemical garbage can.  Dr. Juliet WIlliams of Kymera describes how their company has used modeling and A.I. to design molecular linkers that connect a protein that needs to be degraded with the machinery to tag it for destruction. The goal of this line of therapeutics is to target a suite of proteins that need to be degraded for normal health and development. Their pipeline contains multiple clinical and pre-clinical trials, and the approach is an exciting complement to other drug discovery methods. 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549172v1?rss=1 Authors: Zheng, J.-X., Du, T.-Y., Shao, G.-C., Ma, Z.-H., Jiang, Z.-D., Hu, W., Suo, F., He, W., Dong, M.-Q., Du, L.-L. Abstract: Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. Here we investigate how the poison and antidote products of a fission yeast wtf-family KMD gene can act antagonistically. Both the poison and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs N-terminal PY motifs to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby neutralizing its toxicity and that of the bound poison. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539527v1?rss=1 Authors: Alves Nicolau, C., Renaud, C., Maghe, C., Trillet, K., Jardine, J., Escot, S., David, N. B., Gavard, J., Bidere, N. Abstract: (PCM1) protein, that gravitate as particles around the centrosome. Centriolar satellites have been linked to key cellular processes including the formation of primary cilia and GABARAP-mediated autophagy. However, to date, there is no pharmacological inhibitor of centriolar satellites. Here, we report that necrosulfonamide (NSA), a compound previously shown to target the cell death effector MLKL, induced non-degradative ubiquitination of PCM1. This also caused a defect in ciliogenesis and an accumulation of autophagy markers such as P62 and GABARAPL1, although the overall architecture and distribution of centriolar satellites were maintained. We further found that the depletion of PCM1 lessened the impact of NSA on autophagy. Altogether, NSA may be a valuable tool to study centriolar satellites and provide further insights into their interplay with autophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.530364v1?rss=1 Authors: Berkane, R., Ho-Xuan, H., Glogger, M., Sanz-Martinez, P., Cano-Franco, S., Juretschke, T., Gonzales Cardenas, A., Glaesner, T., Beli, P., Husnjak, K., Doetsch, V., Grumati, P., Heilemann, M., Stolz, A. Abstract: Selective autophagy of the ER (ERphagy) is an important regulator of ER remodeling and critical to maintain cellular homeostasis upon environmental changes. ERphagy receptors link the ER with autophagic membrane thus regulating ERphagy flux. We recently showed that members of the FAM134 family play overlapping and distinct roles during stress-induced ERphagy. Yet the mechanisms on how they are activated remain largely unknown. In this study we analyzed mTOR-mediated dynamic phosphorylation of FAM134 as a trigger of FAM134-driven ERphagy. An unbiased screen of kinase inhibitors revealed CK2 to be essential for FAM134B- and FAM134C-driven ERphagy upon mTOR inhibition. Identified dynamic phosphorylation sites on FAM134C in cells were fitting with predicted CK2 targeting sites, indicating a direct regulatory role of CK2 in FAM134-driven ERphagy. Using super-resolution microscopy, we showed that activity of CK2 is essential for the formation of high-density clusters of FAM134B and FAM134C. Consistently, FAM134B and FAM134C proteins carrying point mutations of selected Serin residues, within their reticulon homology domain, are unable to form high-density clusters. In addition, we provide evidence that the ubiquitination machinery is required for ERphagy and that FAM134B and FAM134C clustering is activated by phospho-dependent ubiquitination. Treatment with CK2 inhibitor SGC-CK2-1 prevents Torin1-induced ERphagy flux as well as ubiquitination of FAM134 proteins and consistently, treatment with E1 inhibitor suppresses Torin1-induced ERphagy flux. Therefore, we propose CK2 dependent phosphorylation of ERphagy receptors precedes ubiquitin-dependent ERphagy flux activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.13.528037v1?rss=1 Authors: Renna, F. J., Steinberg, J. H. E., Manifava, M., Gonzalez, C. D., Tadic, M. S., Orquera, T., Vecino, C. V., Ropolo, A., Rossi, M., Ktistakis, N. T., Vaccaro, M. I. Abstract: Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. VMP1 is an essential autophagy protein whose expression in pancreatic cancer stem cells, carrying activated KRAS, triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches we investigated VMP1 ubiquitination in the autophagic process of human tumor cells. We identified ubiquitination as a post-translational modification for VMP1. VMP1 is ubiquitinated early in autophagosome biogenesis and remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that Cdt2, the substrate recognition subunit of the E3 ligase complex associated with cancer CRL4, is a novel interactor of VMP1. Cdt2 is involved in the VMP1 ubiquitination since Cdt2 relocates from the nucleus to the perinuclear region under VMP1 expression. Moreover, VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with the Cdt2 overexpression. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 in autophagy human tumor cells. VMP1 ubiquitination would be of clinical relevance in tumor cell therapy resistance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526671v1?rss=1 Authors: Luo, H., Todi, S. V., Paulson, H. L., Costa, M. d. C. Abstract: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine (polyQ) in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48 -Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.21.521125v1?rss=1 Authors: Schorova, L., Bedard, N., Khayachi, A., Bolivar-Pedroso, J., Ho, H.-H., Huynh, J., Piccirelli, M., Wang, Y., Plourde, M., Luo, W., Del Cid Pellitero, E., Schlaifer, I., Ye, Y., Durcan, T., Wing, S. S. Abstract: The USP19 deubiquitinase is found in a locus associated with Parkinson's Disease (PD), interacts with heat shock proteins and promotes secretion of a-synuclein (a-syn) through the misfolding associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of a-syn aggregates during the progression of PD, we tested the effect of USP19 knockout (KO) in mice expressing the A53T mutation of a-syn and in whom a-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. The improved pathology was associated with less activation of microglia, higher levels of synaptic marker proteins and improved performance in a tail suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF in the cultured neurons. It also did not affect the propagation of aggregates as assessed by exposing WT or KO neurons to PFF and measuring pSyn positive aggregates in non-exposed adjacent neurons separated using a microfluidics device. We conclude that USP19 instead modulates intracellular dynamics of aggregates. Indeed, at the early time following PFF injection when the number of pSyn positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from primary neurons exposed to PFF with antibodies to ubiquitin or pSyn showed higher levels of ubiquitinated a-syn oligomeric species in the KO neurons. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of a-syn complexes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.08.519265v1?rss=1 Authors: Weinelt, N., Waechtershaeuser, K. N., Smith, S., Andrieux, G., Das, T., Jeiler, B., Roedig, J., Feist, L., Rotter, B., Boerries, M., Pampaloni, F., van Wijk, S. J. L. Abstract: Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endocytosis and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in human cells. Loss of LUBAC activity inhibits necroptosis, without affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Flotillin-1/2 act as putative necroptotic M1 poly-Ub targets that inhibit necroptosis suppression induced by LUBAC inhibition. Finally, we confirm LUBAC-dependent suppression of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as species-specific regulator of necroptosis which prevents MLKL membrane accumulation and pioneer primary human organoids to model necroptosis in near-physiological settings. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.02.506383v1?rss=1 Authors: Ghane, M. A., Allen, Z. D., Miller, C. L., Dong, B., Fang, N., Yang, J. J., Mabb, A. M. Abstract: Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the mechanism by which this occurs and its consequences on other mGluR-mediated signaling events is unknown. Here we show that disrupting Arc ubiquitination on key amino acid residues leads to derangements in Ca2+ release from the endoplasmic reticulum (ER) during pharmacological activation of Group I mGluRs. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination increased Arc self-association and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII. Notably, these interactions were also excluded at secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca2+ signaling that is needed for mGluR-LTD, which, in turn, regulates CaMKII-dependent regulation of Arc. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.22.341784v1?rss=1 Authors: Guo, Y., Chomiak, A. A., Hong, Y., Lowe, C. C., Chan, W.-C., Andrade, J., Pan, H., Zhou, X., Berezovski, E., Monuki, E. S., Feng, Y. Abstract: Aging is an intricate process that is characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. While each of these is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1 associated protein, results in cellular senescence with persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). The high H2Aub initiates proteasome-dependent histone proteolysis, leading to global epigenetic alteration, ubiquitinated protein accumulation, and senescence reinforcement. When these defects occur in mice carrying Brap deletions in cerebral cortical neural progenitors or postnatal neurons, they accelerate brain aging, induce neurodegeneration, and shorten lifespan. As we show H2Aub is also increased in human brain tissues of Alzheimer disease, these data together suggest that chromatin aberrations mediated by H2Aub act as a nexus of multiple aging hallmarks. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.23.218651v1?rss=1 Authors: Steger, M., Ihmor, P., Backman, M., Mueller, S., Daub, H. Abstract: We report a highly optimized proteomics method for in-depth ubiquitination profiling, which combines efficient protein extraction and data-independent acquisition mass spectrometry (DIA-MS). Employing DIA for both spectral library generation and single-shot sample analysis, we quantify up to 70,000 ubiquitinated peptides per MS run with high precision, data completeness and throughput. Our approach resolves the dynamics of ubiquitination and protein degradation with an unprecedented analytical depth. Copy rights belong to original authors. Visit the link for more info

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Guests: Greg Smith and Mark Fuccio Greg Smith joins the TWiVirate to reveal how his lab discovered a switch that controls herpesvirus neuroinvasion, and then we visit the week's news about Zika virus.   Links for this episode Dynamic ubiquitination drives herpesvirus neuroinvasion (PNAS) Releasing Zika virus data in real time (Nature) Zika virus infection and stillbirths (PLoS NTD) Zika virus infection, imported, Italy (Eurosurveillance) Letters read on TWiV 378 This episode is sponsored by 32nd Clinical Virology Symposium and Microbe Magazine Podcast Weekly Science Picks Alan - Science byline counting projectDickson - Bill Nye on climate changeVincent - Zdziarski's blog of things (Apple's motion to vacate)Greg - Atlas, the next generationKathy - Sources of Chaco woodMark - How Dogs Love Us by Gregory Berns Listener Pick Bohdan - The real reason for brains Send your virology questions and comments to twiv@microbe.tv

Guy Salvesen explains how arsenic trioxide treatment switches which SUMO variant is conjugated to an oncoprotein, thus triggering its degradation.

Venuprasad Poojary explains how the ubiquitin ligase Itch prevents chronic skin inflammation.

Vitezslav Bryja, Reinoud de Groot, and Rik Korswagen discovered that Huwe1 ubiquitylates Dishevelled to negatively regulate Wnt signaling.

Michaela Gack discusses how the deubiquitinase USP15 promotes interferon production to prolong the antiviral response.

A deubiquitinase helps control the inflammatory response.

MONDAY, MAY 21 - 2012, 3:30 pm PST/6:30 pm EST Steven Finkbeiner, MD, PhD  Director, Taube-Koret Center for Huntington's Disease Research Senior Investigator and Associate Director, Gladstone Institute of neurological Disease Professor of Neurology and Physiology, University of California, San Francisco Areas of Investigation Research in his laboratory focuses on molecular mechanisms of plasticity and neurodegeneration. A long- term goal of his research is to understand how neuronal activity elicits changes in gene expression that are important for learning and memory and aims to understand how an inherited genetic mutation leads to neuronal dysfunction and degeneration in Huntington's disease (HD). Dr. Finkbeiner will be with us to give us his research updates. Ongoing Studies C-terminus of the NMDA receptor couple Ca2+influx Gene targets of the NMDA receptor Subsynaptic protein translation in learning and memory Polyglutamine expansion degeneration in neurons Ubiquitination and proteasome function in      neurodegeneration Normal function of huntingtin Predictors of neurodegeneration  

Anindya Dutta, Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, USA speaks on "Ubiquitination pathways in the DNA replication and response to DNA damage". This seminar has been recorded by ICGEB Trieste