Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing ...transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation‐based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin‐ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin‐induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full‐genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.
Synopsis
This study presents a novel method for enhancing biotin‐specific signal relative to noise and a complete protein interaction toolbox, which harnesses the power of exogenous biotin ligases in discovering stable and transient protein interactors in yeast.
TurboID is used in baker's yeast for the first time, uncovering transiently interacting protein substrates of the EMC insertase.
ABOLISH is a novel method for reducing endogenous (background) biotinylation and enhancing signal detection from exogenous biotin ligases, such as TurboID.
The BirA‐Avi system is implemented for pairwise protein interaction investigation.
Five whole‐genome libraries are built, in which each yeast protein is N‐terminally tagged with different biotinylation machineries to enable full proteome interaction exploration.
This study presents a novel method for enhancing biotin‐specific signal relative to noise and a complete protein interaction toolbox, which harnesses the power of exogenous biotin ligases in discovering stable and transient protein interactors in yeast.
We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death ...through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.
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•ARTS binds directly to both XIAP and Bcl-2, bringing them into a ternary complex•ARTS bridges between XIAP and Bcl-2, allowing XIAP to serve as an E3-ligase for Bcl-2•ARTS interacts with the BH3 domain of Bcl-2•ARTS functions as a distinct Bcl-2 antagonist by binding and leading to its degradation
Many cancers avoid cell death (apoptosis) by expressing high levels of apoptosis inhibitors, such as Bcl-2. Thus, Bcl-2 is a major target for cancer therapy. Edison et al. describe a mechanism by which the ARTS protein promotes proteasome-mediated degradation of Bcl-2 and thereby stimulates cell death.
The cell nucleus is constantly subjected to externally applied forces. During metazoan evolution, the nucleus has been optimized to allow physical deformability while protecting the genome under ...load. Aberrant nucleus mechanics can alter cell migration across narrow spaces in cancer metastasis and immune response and disrupt nucleus mechanosensitivity. Uncovering the mechanical roles of lamins and chromatin is imperative for understanding the implications of physiological forces on cells and nuclei. Lamin‐knockout and ‐rescue fibroblasts and probed nucleus response to physiologically relevant stresses are generated. A minimal viscoelastic model is presented that captures dynamic resistance across different cell types, lamin composition, phosphorylation states, and chromatin condensation. The model is conserved at low and high loading and is validated by micropipette aspiration and nanoindentation rheology. A time scale emerges that separates between dominantly elastic and dominantly viscous regimes. While lamin‐A and lamin‐B1 contribute to nucleus stiffness, viscosity is specified mostly by lamin‐A. Elastic and viscous association of lamin‐B1 and lamin‐A is supported by transcriptional and proteomic profiling analyses. Chromatin decondensation quantified by electron microscopy softens the nucleus unless lamin‐A is expressed. A mechanical framework is provided for assessing nucleus response to applied forces in health and disease.
The cell nucleus is evolutionarily‐optimized to deform while protecting the genome under load. A viscoelastic model of nucleus resistance to physiologically relevant stresses that is conserved across cell types and nucleoskeletal perturbations is presented. An inherent timescale separates between elastic stretching set by lamin‐A and viscoelastic fluid deformation. Both A‐ and B‐type lamins contribute to steady‐state stiffness whereas lamin‐A and chromatin decondensation increase viscosity.
Protection from viral infections depends on immunoglobulin isotype switching, which endows antibodies with effector functions. Here, we find that the protein kinase DYRK1A is essential for B ...cell-mediated protection from viral infection and effective vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells are impaired in CSR activity in vivo and in vitro. Phosphoproteomic screens and kinase-activity assays identify MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site impaired CSR. After CSR and germinal center (GC) seeding, DYRK1A is required for attenuation of B cell proliferation. These findings demonstrate DYRK1A-mediated biological mechanisms of B cell immune responses that may be used for therapeutic manipulation in antibody-mediated autoimmunity.
The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome ...complex (CSN) through both enzymatic (deneddylation) and nonenzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.
The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains ...unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
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•Linker histone H1 is post-translationally SUMOylated in ESCs•SUMOylation of linker histone H1 drives its fixation onto condensed heterochromatin•Loss of SUMOylated H1 is associated with chromatin decompaction in ESCs•SUMOylation of H1 represses the reactivation of the totipotency program in ESCs
Sheban et al. demonstrate that linker histone H1 is modified by SUMO to modulate the chromatin landscape and repress the totipotency program in ESCs. This process, in turn, ensures proper determination of early embryonic cell fate.
BACKGROUNDCytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal ...care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury.METHODSWe performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays.RESULTSUsing unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins - the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3-binding protein (Gal-3BP) - were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%-93.8% positive predictive value, and 92.9%-92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSIONAnalysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.FUNDINGIsrael Science Foundation (530/18 and IPMP 3432/19); Research Fund - Hadassah Medical Organization.
Despite being the second most common neurodegenerative disorder, little is known about Parkinson's disease (PD) pathogenesis. A number of genetic factors predispose towards PD, among them mutations ...in GBA1, which encodes the lysosomal enzyme acid-β-glucosidase. We now perform non-targeted, mass spectrometry based quantitative proteomics on five brain regions from PD patients with a GBA1 mutation (PD-GBA) and compare to age- and sex-matched idiopathic PD patients (IPD) and controls. Two proteins were differentially-expressed in all five brain regions whereas significant differences were detected between the brain regions, with changes consistent with loss of dopaminergic signaling in the substantia nigra, and activation of a number of pathways in the cingulate gyrus, including ceramide synthesis. Mitochondrial oxidative phosphorylation was inactivated in PD samples in most brain regions and to a larger extent in PD-GBA. This study provides a comprehensive large-scale proteomics dataset for the study of PD-GBA.
White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for the ...chemical industry due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative-directed evolution protocols have been applied to improve their expression, activity, and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express. First, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib-designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3–4 subtle mutations that change the polarity, solvation, and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.
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