23 January 2020

Heat shock factor 1 (HSF1) regulates cellular adaptation to challenges such as heat shock and oxidative and proteotoxic stresses. We have recently reported a previously unappreciated role for HSF1 in the regulation of energy metabolism in fat tissues; however, whether HSF1 is differentially expressed in adipose depots and how its levels are regulated in fat tissues remain unclear. Here, we show that HSF1 levels are higher in brown and subcutaneous fat tissues than in those in the visceral depot and that HSF1 is more abundant in differentiated, thermogenic adipocytes. Gene expression experiments indicated that HSF1 is transcriptionally regulated in fat by agents that modulate cAMP levels, by cold exposure, and by pharmacological stimulation of β-adrenergic signaling. An in silico promoter analysis helped identify a putative response element for activating transcription factor 3 (ATF3) at −258 to −250 base pairs from the HSF1 transcriptional start site, and electrophoretic mobility shift and ChIP assays confirmed ATF3 binding to this sequence. Furthermore, functional assays disclosed that ATF3 is necessary and sufficient for HSF1 regulation. Detailed gene expression analysis revealed that ATF3 is one of the most highly induced ATFs in thermogenic tissues of mice exposed to cold temperatures or treated with the β-adrenergic receptor agonist CL316,243 and that its expression is induced by modulators of cAMP levels in isolated adipocytes. To the best of our knowledge, our results show for the first time that HSF1 is transcriptionally controlled by ATF3 in response to classic stimuli that promote heat generation in thermogenic tissues.

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Charles Collot, Frank Merle and Pierre Raphaël
J. Amer. Math. Soc. 33 (2020), 527-607.
Abstract, references and article information

Roland Bruderer
May 1, 2015; 14:1400-1410
Research

Amelia C. Peterson
Nov 1, 2012; 11:1475-1488
Technological Innovation and Resources

Mitsunori Fukuda
Jun 1, 2008; 7:1031-1042
Research

Hasmik Keshishian
Dec 1, 2007; 6:2212-2229
Research

Qiang Tian
Oct 1, 2004; 3:960-969
Research

Ludovic C. Gillet
Jun 1, 2012; 11:O111.016717-O111.016717
Research

Martin R. Larsen
Jul 1, 2005; 4:873-886
Technology

Eiji Kinoshita
Apr 1, 2006; 5:749-757
Technology

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

16 January 2020

Invitation Only Research Event

4 March 2020 , Volume 96, Number 2

4 March 2020 , Volume 96, Number 2

Members Event

Event participants

Oded Haklai, Associate Professor, Department of Political Studies, Queen's University, Ontario
Neophytos Loizides, Reader, University of Kent; Leverhulme Trust Research Fellow
Madurika Rasaratnam, Lecturer, International Conflict Analysis, University of Kent
Chair: Evangelos Liaras, IE University, Madrid; Academy Associate, Chatham House

10 August 2016

James M. Ntambi
Sep 1, 1999; 40:1549-1558
Reviews

EJ Blanchette-Mackie
Jun 1, 1995; 36:1211-1226
Articles

JF Oram
Dec 1, 1996; 37:2473-2491
Reviews

DL Brasaemle
Nov 1, 1997; 38:2249-2263
Articles

K Schoonjans
May 1, 1996; 37:907-925
Reviews

Invitation Only Research Event

Event participants

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (~3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed “templated folding,” whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Members Event

Event participants

Dr Marja Lehto, Special Rapporteur, International Law Commission, UN
Doug Weir, Research and Policy Director, The Conflict and Environment Observatory
Chair: Elizabeth Wilmshurst CMG, Distinguished Fellow, International Law Programme, Chatham House
 

14 November 2019

Research Event