Biology subjects

Nilsson, J.

Publications and source records attributed to Nilsson, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting the MCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additional regulatory components within the SAC and establish a novel method to interrogate Cdc20 function.

cell biology↗

bioRxiv2023

Global substrate identification and high throughput in vitro dephosphorylation reactions uncover PP1 and PP2A-B55 specificity principles

Phosphoprotein phosphatases (PPPs) dephosphorylate Serine (Ser)/Threonine (Thr) residues to regulate major signaling pathways and cellular transitions. Despite the central role of PPPs the substrates in most cellular processes and the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A- B55, which reveals common and unique preferences for the two phosphatases. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE:Dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit, resulting in defective cytokinesis thus underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and demostrate how binding of phosphatases to substrates shapes dephosphorylation specificity. Collectively, we develop novel approaches to advance our ability to investigate protein phosphatases and use these to provide a framework for understanding mitotic exit regulation by dephosphorylation.

cell biology↗

bioRxiv2023

Group 2 innate lymphoid cells constrain type 3/17 lymphocytes in shared stromal niches to restrict liver fibrosis

Group 2 innate lymphoid cells (ILC2s) cooperate with adaptive Th2 cells as key organizers of tissue type 2 immune responses, while a spectrum of innate and adaptive lymphocytes coordinate early type 3/17 immunity. Both type 2 and type 3/17 lymphocyte associated cytokines are linked to tissue fibrosis, but how their dynamic and spatial topographies may direct beneficial or pathologic organ remodelling is unclear. Here we used volumetric imaging in models of liver fibrosis, finding accumulation of periportal and fibrotic tract IL-5+ lymphocytes, predominantly ILC2s, in close proximity to expanded type 3/17 lymphocytes and IL-33high niche fibroblasts. Ablation of IL-5+ lymphocytes worsened carbon tetrachloride-and bile duct ligation-induced liver fibrosis with increased niche IL-17A+ type 3/17 lymphocytes, predominantly {gamma}{delta} T cells. In contrast, concurrent ablation of IL-5+ and IL-17A+ lymphocytes reduced this progressive liver fibrosis, suggesting a cross-regulation of type 2 and type 3 lymphocytes at specialized fibroblast niches that tunes hepatic fibrosis.

immunology↗

bioRxiv2022

Fate and plasticity of SARS-CoV-2-specific B cells during memory and recall response in humans

B cell responses to different pathogens recruit tailored effector mechanisms, resulting in functionally specialized subsets. For human memory B cells (MBCs), these include CD21+ resting, CD21-CD27+ activated, and CD21-CD27- atypical cells. Whether these subsets follow deterministic or interconnected fates is unknown. We demonstrate in COVID-19 patients that single clones of SARS-CoV-2-specific MBCs followed multiple fates with distinctive phenotypic and functional characteristics. 6-12 months after infection, most circulating MBCs were CD21+ resting cells, which also accumulated in peripheral lymphoid organs where they acquired markers of tissue residency. Conversely, at acute infection and following SARS-CoV-2-specific immunization, CD21- MBCs became the predominant subsets, with atypical MBCs expressing high T-bet, inhibitory molecules, and distinct chemokine receptors. B cell receptor sequencing allowed tracking of individual MBC clones differentiating into CD21+, CD21-CD27+, and CD21-CD27- cell fates. Collectively, single MBC clones can adopt functionally different trajectories, thus contributing to immunity to infection.

immunology↗

bioRxiv2022

Large-scale identification of phospho-modulated motif-based protein-protein interactions

Phosphorylation is an extensively studied post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Deciphering which of the hundreds of thousands of phosphosites in the proteome that regulate interactions remains challenging. We generated a proteomic peptide-phage display (ProP-PD) library to screen for phosphosites that regulate short linear motif-based interactions. The phage peptidome covers 13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome, each phosphosite being represented as a wildtype and a phosphomimetic variant. We screened 73 modular domains and identified 252 putative phospho-modulated interactions. Affinity measurements confirmed the phosphomodulation of 16 out of 21 tested interactions. We discovered a novel phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP). We validated the phospho-dependent clathrin interaction in a cellular context and found it to be essential for the mitotic function of HURP. Structural characterisation elucidated the molecular basis for the phospho-dependency of the clathrin-HURP interaction. Collectively, our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated SLiM-based interactions required for cellular function.

biochemistry↗

bioRxiv2022

Separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogues with Bub1 being a kinase while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can supports chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation resulting in reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

cell biology↗

bioRxiv2022

A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis

TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.

cell biology↗

bioRxiv2021

Generation and diversification of recombinant monoclonal antibodies for studying mitosis

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

cell biology↗

bioRxiv2021

CD8+ T cell signature in acute SARS-CoV-2 infection identifies memory precursors

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing coronavirus disease 19 (COVID-19) pandemic, a key question has focused on whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor (TCR) sequencing we longitudinally characterize individual SARS-CoV-2-specific CD8+ T cells of COVID-19 patients from acute infection to one year into recovery and find a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting one year after acute infection re-express CD45RA and interleukin-7 receptor (CD127), upregulate T cell factor-1 (TCF1), and maintain low CCR7, thus resembling CD45RA+ effector-memory T (TEMRA) cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones giving rise to long-lived cells, whereas prolonged proliferation and mammalian target of rapamycin (mTOR) signaling are associated with clone contraction and disappearance. Collectively, we identify a transcriptional signature differentiating short-from long-lived memory CD8+ T cells following an acute virus infection in humans.

immunology↗

bioRxiv2021

Cellular toxicity of iHAP1 and DT-061 does not occur through PP2A-B56 targeting

PP2A is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The small molecule compounds iHAP1 and DT-061 have recently been reported by Leonard et al. (2020) and Morita et al. (2020) in Cell to selectively stabilize specific PP2A-B56 complexes which mediate cell killing. Here, we show that this is not the case and question key findings in these papers. Through genome wide CRISPR-Cas9 screens, we uncover the biological pathways targeted by these compounds. We find that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the endoplasmic reticulum and we directly visualize DT-061 in cytoplasmic granules that co-localize with Golgi markers. Our work demonstrates that iHAP1 and DT-061 cannot be used for dissecting PP2A-B56 biology.

cell biology↗

bioRxiv2021

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, current methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a scalable viral peptide discovery approach covering 229 RNA viruses that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an {Phi}xFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its {Phi}xFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction blocks SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.

biochemistry↗

bioRxiv2021

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by promoting RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

cell biology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

A highly conserved binding pocket on PP2A-B56 is required for shugoshin binding and cohesion protection

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human Sgo1 to the PP2A-B56 phosphatase through a coiled coil (CC) region is believed to mediate cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-Sgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket of the B56 regulatory subunit is required for Sgo1 binding and cohesion protection. Consistent with this, we show that Sgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. Surprisingly, we identify B56 and Sgo1 mutants that prevent complex formation yet support cohesion protection and normal mitotic progression. This suggests that Sgo1 and PP2A-B56 have cohesion protection activity independently of complex formation. Collectively our work provides important insight into cohesion protection during mitosis.

cell biology↗

bioRxiv2020

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

cell biology↗

bioRxiv2020

Revealing interspecies transmission barriers of avian influenza A viruses

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further to mammals including humans. Investigating molecular virus-host interactions dictating this process and the adaptations to the new hosts that follow is vital to understand zoonotic IAV spread. Receptor incompatibility has been suggested to limit zoonotic IAV transmission from the wild bird reservoir. Other barriers to interspecies transmission, particularly within the avian system, largely remain elusive. Through assessment of infection dynamics of mallard origin IAV in two different avian hosts, coupled with studies of receptor expression and host response we aimed to reveal the host-pathogen interactions in a cross-species transmission event. We found that shedding patterns and innate immune responses were highly dependent on viral genotypes, host species and inoculation routes, but less dependent on receptor expression. Further, in contrary to the prevailing dogma we demonstrate that birds can produce a wide range of different sialylated structures also found in mammals, e.g. extended N- and O-linked Neu5Ac2,6 terminated glycans. Overall, receptor incompatibility is not the sole transmission barrier for IAV between birds and to humans, but other host-pathogen factors deserve dedicated studies to achieve proper pandemic preparedness.

microbiology↗

bioRxiv2020

A distinct innate immune signature marks progression from mild to severe COVID-19

Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. Excessive inflammation has been postulated to be a major factor in the pathogenesis of severe COVID-19 and innate immune mechanisms are likely to be central in the inflammatory response. We used 40-plex mass cytometry and targeted serum proteomics to profile innate immune cell populations from peripheral blood of patients with mild or severe COVID-19 and healthy controls. Sampling at different stages of COVID-19 allowed us to reconstruct a pseudo-temporal trajectory of the innate immune response. Despite the expected patient heterogeneity, we identified consistent changes during the course of the infection. A rapid and early surge of CD169+ monocytes associated with an IFN{gamma}+MCP-2+ signature quickly followed symptom onset; at symptom onset, patients with mild and severe COVID-19 had a similar signature, but over the course of the disease, the differences between patients with mild and severe disease increased. Later in the disease course, we observed a more pronounced re-appearance of intermediate/non-classical monocytes and mounting systemic CCL3 and CCL4 levels in patients with severe disease. Our data provide new insights into the dynamic nature of the early inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identifies sustained pathological innate immune responses as a likely key mechanism in severe COVID-19, further supporting investigation of targeted anti-inflammatory interventions in severe COVID-19.

immunology↗

bioRxiv2020

Lymphopenia-induced T cell proliferation is a hallmark of severe COVID-19

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a broad clinical presentation ranging from asymptomatic infection to fatal disease. Different features associated with the immune response to SARS-CoV-2, such as hyperinflammation and reduction of peripheral CD8+ T cell counts are strongly associated with severe disease. Here, we confirm the reduction in peripheral CD8+ T cells both in relative and absolute terms and identify T cell apoptosis and migration into inflamed tissues as possible mechanisms driving peripheral T cell lymphopenia. Furthermore, we find evidence of elevated serum interleukin-7, thus indicating systemic T cell paucity and signs of increased T cell proliferation in patients with severe lymphopenia. Following T cell lymphopenia in our pseudo-longitudinal time course, we observed expansion and recovery of poly-specific antiviral T cells, thus arguing for lymphopenia-induced T cell proliferation. In summary, this study suggests that extensive T cell loss and subsequent T cell proliferation are characteristic of severe COVID-19.

immunology↗