Biology subjects

Dikic, I.

Publications and source records attributed to Dikic, I..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Stochastic modeling of the dynamics of Salmonella infection of epithelial cells

Bacteria of the Salmonella genus are intracellular pathogens, which cause gastroenteritis and typhoid fever in animals and humans, and are responsible for millions of infections and thousands of deaths across the world every year. Furthermore, Salmonella has played the role of a model organism for studying host-pathogen interactions. Taking these two aspects into account, enormous efforts in the literature are devoted to study this intracellular pathogen. Within epithelial cells, there are two distinct subpopulations of Salmonella: (i) a large fraction of Salmonella, which are enclosed by vacuoles, and (ii) a small fraction of hyper-replicating cytosolic Salmonella. Here, by considering the infection of epithelial cells by Salmonella as a discrete-state, continuous-time Markov process, we propose a stochastic model of infection, which includes the invasion of Salmonella into the epithelial cells by a cooperative strategy, the replication inside the Salmonella-containing vacuole, and the bacterial proliferation in the cytosol. The xenophagic degradation of cytosolic bacteria is considered, too. The stochastic approach provides important insights into stochastic variation and heterogeneity of the vacuolar and cytosolic Salmonella populations on a single-cell level over time. Specifically, we predict the percentage of infected human epithelial cells depending on the incubation time and the multiplicity of infection, an d the bacterial load of the infected cells at different post-infection times.

systems biology↗

bioRxiv2023

An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

Targeted protein degradation is a pharmacological modality based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via bifunctional compounds (PROTACs) composed of two separate warheads that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Using orthogonal genetic screening, biophysical characterization, and structural reconstitution, we investigate the mode of action of bifunctional BRD2/4 degraders (IBG1-4) and find that - instead of connecting target and ligase in trans as PROTACs do - they simultaneously engage two adjacent domains of the target protein in cis. This conformational change glues BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities which, albeit pre-existing, do not translate to BRD4 degradation in absence of compound. Structural insights into the ternary BRD4:IBG1:DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, termed intramolecular bivalent glues (IBGs), which work by bridging protein domains to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

biochemistry↗

bioRxiv2023

Stabilisation of β-Catenin-WNT signalling by USP10 in APC-truncated colorectal cancer drives cancer stemness and enables super-competitor signalling

The contribution of deubiquitylating enzymes to {beta}-Catenin stabilisation in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, we report the deubiquitylase USP10 as an APC-truncation- specific enhancer of {beta}-Catenin stability, potentiating WNT signalling in CRC and cancer stem cells. Mechanistically, interaction studies in various CRC cell lines and in vitro binding studies, together with computational modelling, revealed that USP10 binding to {beta}-Catenin is mediated via the unstructured N-terminus of USP10 and requires the absence of full-length APC. Notably, loss of USP10 in CRISPR engineered intestinal organoids reduces tumorigenic properties of CRC and blocks the super competitor-signalling of APC-mutated CRC. Furthermore, reduction of USP10 induces the expression of differentiation genes, and opposes the APC-truncated phenotype in an intestinal hyperplasia model of D.melanogaster. Taken together, our findings reveal USP10s role in intestinal tumourigenesis by stabilising {beta}-Catenin, leading to aberrant WNT signalling, enhancing cancer cell stemness and implicate the DUB USP10 as a cancer specific therapeutic vulnerability in Apc truncated CRC.

cancer biology↗

bioRxiv2022

TBC1D15 potentiates lysosomal regeneration from damaged membranes

Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in TFEB/TFE3-depleted cells. We combined proximity labelling based proteomics, biochemistry and high-resolution microscopy to unravel a new lysosomal membrane regeneration pathway which is dependent on ATG8, lysosomal membrane protein LIMP2, the Rab7 GAP TBC1D15, and proteins required for autophagic lysosomal reformation (ALR) including Dynamin2, Kinesin5B and Clathrin. Upon lysosomal damage, LIMP2 act as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 hydrolyses Rab7-GTP to segregate the damaged lysosomal mass and provides a scaffold to assemble and stabilize the ALR machinery, potentiating the formation of lysosomal tubules and subsequent Dynamin2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.

cell biology↗

bioRxiv2022

mTOR inhibitor-independent Autophagy Activator Ameliorates Cellular Tauopathy and Prionopathy Neurodegeneration Phenotypes

Autophagy-lysosomal impairment is an early and prominent feature of neurodegeneration. Autophagy activation reduces protein aggregates and lipid level abnormalities. We performed a high-content imaging-based screen assessing 940,000 small molecules to identify those that reduce lipid droplet numbers. Of 77 validated, structurally diverse hits, 24 increased autophagy flux reporter activity, consistent with accelerated lipid droplet clearance by lipophagy. Of these, we show that CCT020312 activates autophagy independently of mammalian target of rapamycin (mTOR) inhibition, to avoid immunosuppression. CCT020312 reduced insoluble phosphorylated tau levels and tau-mediated neuronal stress vulnerability, as well as reducing intracellular A{beta} levels within directly induced neurons bearing epigenetic marks of aging derived from Alzheimers patient fibroblasts. Moreover, CCT020312 cleared mutant prion protein aggregates and normalized trafficking deficiencies in axons of a cellular model of familial prion disease. Autophagy is widely considered a promising strategy to attenuate neurodegeneration, and here we introduce a strategy to discover new pharmacology.

cell biology↗

bioRxiv2021

Mathematical modeling of the molecular switch of TNFR1-mediated signaling pathways using Petri nets

The paper describes a mathematical model of the molecular switch of cell survival, apoptosis, and necroptosis in cellular signaling pathways initiated by tumor necrosis factor 1. Based on experimental findings in the current literature, we constructed a Petri net model in terms of detailed molecular reactions for the molecular players, protein complexes, post-translational modifications, and cross talk. The model comprises 118 biochemical entities, 130 reactions, and 299 connecting edges. Applying Petri net analysis techniques, we found 279 pathways describing complete signal flows from receptor activation to cellular response, representing the combinatorial diversity of functional pathways.120 pathways steered the cell to survival, whereas 58 and 35 pathways led to apoptosis and necroptosis, respectively. For 65 pathways, the triggered response was not deterministic, leading to multiple possible outcomes. Based on the Petri net, we investigated the detailed in silico knockout behavior and identified important checkpoints of the TNFR1 signaling pathway in terms of ubiquitination within complex I and the gene expression dependent on NF-{kappa}B, which controls the caspase activity in complex II and apoptosis induction.

systems biology↗

bioRxiv2021

USP28 enables oncogenic transformation of respiratory cells and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and {Delta}NP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a pre-malignant state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.

cancer biology↗

bioRxiv2021

FAM134B-RHD Protein Clustering Drives Spontaneous Budding of Asymmetric Membranes

Living cells constantly remodel the shape of their lipid membranes. In the endo-plasmic reticulum (ER), the reticulon homology domain (RHD) of the reticulophagy regulator 1 (RETR1/FAM134B) forms dense autophagic puncta that are associated with membrane removal by ER-phagy. In molecular dynamics (MD) simulations, we find that FAM134B-RHD spontaneously forms clusters, driven in part by curvature-mediated attraction. At a critical size, the FAM134B-RHD clusters induce the formation of membrane buds. The kinetics of budding depends sensitively on protein concentration and bilayer asymmetry. Our MD simulations shed light on the role of FAM134B-RHD in ER-phagy and show that membrane asymmetry can be used to modulate the kinetics barrier for membrane remodeling. Graphical TOC Entry O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2020

Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

biochemistry↗

bioRxiv2020

Proteomics of autophagy deficient macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

Defective autophagy is associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohns disease by enhancing innate immunity in macrophages. However, autophagy also mediates clearance of intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in antimicrobial immunity. In this study, we found that loss of Atg16l1 in macrophages enhanced the killing of virulent Shigella flexneri (S.flexneri), an enteric bacterium that resides within the cytosol by escaping all membrane-bound compartments. Quantitative multiplexed proteomics revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which also promoted S.flexneri killing. Consistently, myeloid cell-specific deletion of Atg16l1 accelerated bacterial clearance in vivo. Finally, pharmacological modulation of oxidative stress by suppression of cysteine import conferred enhanced microbicidal properties to wild type macrophages. These findings demonstrate that control of oxidative stress by ATG16L1 regulates antimicrobial immunity against intracellular pathogens. Impact statementMaculins et al utilize multiplexed mass spectrometry to show that loss of the autophagy gene Atg16l1 in macrophages enhances antimicrobial immunity against intracellular pathogens via the oxidative stress response.

cell biology↗

bioRxiv2020

Inhibition of USP28 overcomes Cisplatin-Resistance of Squamous Tumors by Suppression of the Fanconi Anemia Pathway.

Squamous cell carcinomas (SCC) frequently have a limited response to or develop resistance to platinum-based chemotherapy, and have an exceptionally high tumor mutational burden. As a consequence, overall survival is limited and novel therapeutic strategies are urgently required, especially in light of a rising incidences. SCC tumors express {Delta}Np63, a potent regulator of the Fanconi Anemia (FA) DNA-damage response pathway during chemotherapy, thereby directly contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 affects the FA DNA repair pathway during cisplatin treatment in SCC, thereby influencing therapy outcome. In an ATR-dependent fashion, USP28 is phosphorylated and activated to positively regulate the DNA damage response. Inhibition of USP28 reduces recombinational repair via an {Delta}Np63-Fanconi Anemia pathway axis, and weakens the ability of tumor cells to accurately repair DNA. Our study presents a novel mechanism by which tumor cells, and in particular {Delta}Np63 expressing SCC, can be targeted to overcome chemotherapy resistance. SignificanceLimited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor {Delta}Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-{Delta}Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.

cancer biology↗

bioRxiv2020

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the secretory pathway, including cytokine release in cells. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

cell biology↗

bioRxiv2020

Combinatorial CRISPR screening reveals functional buffering in autophagy

Functional genomics studies in model organisms and human cell lines provided important insights into gene functions and their context-dependent role in genetic circuits. However, our functional understanding of many of these genes and how they combinatorically regulate key biological processes, remains limited. To enable the SpCas9-dependent mapping of gene-gene interactions in human cells, we established 3Cs multiplexing for the generation of combinatorial gRNA libraries in a distribution-unbiased manner and demonstrate its robust performance. The optimal number for combinatorial hit calling was 16 gRNA pairs and the skew of a librarys distribution was identified as a critical parameter dictating experimental scale and data quality. Our approach enabled us to investigate 247,032 gRNA-pairs targeting 12,736 gene-interactions in human autophagy. We identified novel genes essential for autophagy and provide experimental evidence that gene-associated categories of phenotypic strengths exist in autophagy. Furthermore, circuits of autophagy gene interactions reveal redundant nodes driven by paralog genes. Our combinatorial 3Cs approach is broadly suitable to investigate unexpected gene-interaction phenotypes in unperturbed and diseased cell contexts.

cell biology↗

bioRxiv2020

Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

microbiology↗

bioRxiv2020

Novel class of OTU deubiquitinases regulate substrate ubiquitinationupon Legionella infection

Legionella pneumophila is a gram-negative pathogenic bacterium that causes Legionaries disease. The Legionella genome codes more than 300 effector proteins able to modulate host-pathogen interactions during infection. Among them are also enzymes altering the host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on homology-detection screening on 305 Legionella effector proteins, we identified two Legionella OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was determined at 2.4 [A] and compared with other OTU deubiquitinases, including LotB. Unlike the classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an extended helical lobe between the Cys-loop and the variable loop, which define a novel class of OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC interact with ubiquitin. LotB has an additional ubiquitin binding site (S1) enabling specific cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC identified different categories of host-substrates for these two related DUBs. Together, our results provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of bacterial deubiquitinases in host-pathogen interactions.

molecular biology↗

bioRxiv2019

The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

cell biology↗

bioRxiv2019

Wss1 promotes replication stress tolerance by degrading histones.

Timely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions.

cell biology↗

bioRxiv2019

TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease

Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autoph-agosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.

biochemistry↗