MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.This work was funded by the VW foundation (Life?, #93089, #93092, #93090) to RE, MS, and JS, by the Deutsche Forschungsgemeinschaft in the framework of the SFB894 to RE and the SFB1027 to both JH and RE, and by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 866011) to RE. MS is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics

Janus-kinaasien molekyylinsisäinen säätely

Janus-kinaasit (JAKit) ovat ei-reseptorisia tyrosiinikinaaseja, jotka välittävät yli 60 sytokiinin viestejä soluissa. Nämä viestit säätelevät lukuisia biologisia tapahtumia, kuten immuunijärjestelmän toimintaa, hematopoieesia, aineenvaihduntaa sekä kehitystä. JAKit ovat monidomeenisia proteeineja, joissa viestivää, aktiivista tyrosiinikinaasidomeenia (JH1) edeltää nk. pseudokinaasidomeeni (JH2). JH2 on ensisijaisen tärkeä JAK-aktiivisuuden säätelyssä. Domeenista onkin löydetty lukuisia kliinisesti merkittäviä mutaatioita, kuten autosomaalisia JAK2-mutaatioita, jotka aiheuttavat ligandiriippumatonta JAK-aktivaatiota ja siten johtavat hematopoieettisiin maligniteetteihin. Tässä työssä esitellyissä tutkimuksissa selvitettiin JH2:n sekä sen mutaatioiden toimintaa JAK-säätelyssä. Tutkimuksissa havaittiin, että kaikissa JAK JH2:ssa on toiminnallinen nukleotidinsitomistasku, joka kykenee sitomaan ATP:ta ja pienmolekyylisiä inhibiittoreita. Tärkeimpänä löydöksenä havaittiin, että ATP:n sitoutumisen estäminen JAK2 JH2:n ATP-sitomistaskuun alentaa ligandiriippumatonta JAK2-aktivaatiota. Nämä havainnot osoittavat, että JAK2 JH2 on mahdollinen lääkekohde kohdennettujen JAK-inhibiittoreiden kehittämiselle. Tällaiset inhibiittorit, jotka estäisivät kohdennetusti mutatoituneen (muttei villityyppisen) JAKin toimintaa, olisivat merkittävä parannus verrattuna nykyisiin JAK-estäjiin, jotka eivät kykene erottelemaan villityyppisen ja mutatoituneen JAKin välillä, eivätkä siten pysty parantamaan tautia. Tutkimuksissa kehitettiin yhteistyöprojektina myös molekyylimalli JH2:n toiminnasta JH1:n aktiivisuuden säätelijänä. JH2–JH1-malli selittää useimpien tunnettujen kliinisten JAK2-mutaatioiden toiminnan molekyylitasolla. Lisäksi suoritettiin systemaattinen analyysi JAK2-mutaatioista, jotka voivat estää tautia aiheuttavan JAK2-hyperaktivaation. Tämä analyysi tarkentaa ymmärrystämme JAKien toiminnasta sytokiinivälitteisessä sekä sytokiineista riippumattomassa JAK-aktivaatiossa ja samalla tunnistaa aiemmin tuntemattoman JAK2 JH2 rajapinnan, joka on välttämätön interferoni-γ signaloinnille.Janus kinases (JAKs) are non-receptor tyrosine kinases that mediate signalling of around sixty different cytokines governing various biological processes from the regulation of the immune system, to control of haematopoiesis, metabolism, and development. JAKs are multidomain proteins, in which the tyrosine kinase domain (JH1) is preceded by a pseudokinase domain (JH2). JH2 has critical regulatory functions and is a hotspot for many known oncogenic driver mutations. These mutations, which cause ligand-independent JAK activation, underlie various diseases—most notably haematopoietic malignancies caused by somatic JAK2 JH2 mutations. In the work presented here, we analysed the functions of JH2 and its mutations in the regulation of JAK activity. We found that all JAK JH2s have functional nucleotide-binding sites accessible to ATP and small molecule inhibitors. Most importantly, we found that disruption of ATP binding to JAK2 JH2 suppresses ligand-independent activation, thus identifying the JAK2 JH2 ATP-binding site as a potential drug target for the development of mutation-specific inhibitors. These inhibitors would be a distinct improvement over inhibitors of JAK2 currently used to treat MPNs, as current inhibitors do not distinguish between mutated and wild-type JAK2, and are unable to eradicate the disease. We also present a collaboration effort leading to a simulation-based model for JH2-mediated inhibition of JH1, thereby providing rationale for most known clinical JAK2 mutations. Moreover, we refine our understanding of ligand-mediated and ligand-independent activation of JAKs by presenting a systematic analysis of JAK2 mutations capable of inhibiting ligand-independent hyperactivation. We further identify a novel interface in JAK2 JH2, which is needed for heteromeric JAK2 activation in interferon-γ signalling

Janus-kinaasien molekyylinsisäinen säätely

Janus-kinaasit (JAKit) ovat ei-reseptorisia tyrosiinikinaaseja, jotka välittävät yli 60 sytokiinin viestejä soluissa. Nämä viestit säätelevät lukuisia biologisia tapahtumia, kuten immuunijärjestelmän toimintaa, hematopoieesia, aineenvaihduntaa sekä kehitystä. JAKit ovat monidomeenisia proteeineja, joissa viestivää, aktiivista tyrosiinikinaasidomeenia (JH1) edeltää nk. pseudokinaasidomeeni (JH2). JH2 on ensisijaisen tärkeä JAK-aktiivisuuden säätelyssä. Domeenista onkin löydetty lukuisia kliinisesti merkittäviä mutaatioita, kuten autosomaalisia JAK2-mutaatioita, jotka aiheuttavat ligandiriippumatonta JAK-aktivaatiota ja siten johtavat hematopoieettisiin maligniteetteihin. Tässä työssä esitellyissä tutkimuksissa selvitettiin JH2:n sekä sen mutaatioiden toimintaa JAK-säätelyssä. Tutkimuksissa havaittiin, että kaikissa JAK JH2:ssa on toiminnallinen nukleotidinsitomistasku, joka kykenee sitomaan ATP:ta ja pienmolekyylisiä inhibiittoreita. Tärkeimpänä löydöksenä havaittiin, että ATP:n sitoutumisen estäminen JAK2 JH2:n ATP-sitomistaskuun alentaa ligandiriippumatonta JAK2-aktivaatiota. Nämä havainnot osoittavat, että JAK2 JH2 on mahdollinen lääkekohde kohdennettujen JAK-inhibiittoreiden kehittämiselle. Tällaiset inhibiittorit, jotka estäisivät kohdennetusti mutatoituneen (muttei villityyppisen) JAKin toimintaa, olisivat merkittävä parannus verrattuna nykyisiin JAK-estäjiin, jotka eivät kykene erottelemaan villityyppisen ja mutatoituneen JAKin välillä, eivätkä siten pysty parantamaan tautia. Tutkimuksissa kehitettiin yhteistyöprojektina myös molekyylimalli JH2:n toiminnasta JH1:n aktiivisuuden säätelijänä. JH2–JH1-malli selittää useimpien tunnettujen kliinisten JAK2-mutaatioiden toiminnan molekyylitasolla. Lisäksi suoritettiin systemaattinen analyysi JAK2-mutaatioista, jotka voivat estää tautia aiheuttavan JAK2-hyperaktivaation. Tämä analyysi tarkentaa ymmärrystämme JAKien toiminnasta sytokiinivälitteisessä sekä sytokiineista riippumattomassa JAK-aktivaatiossa ja samalla tunnistaa aiemmin tuntemattoman JAK2 JH2 rajapinnan, joka on välttämätön interferoni-γ signaloinnille.Janus kinases (JAKs) are non-receptor tyrosine kinases that mediate signalling of around sixty different cytokines governing various biological processes from the regulation of the immune system, to control of haematopoiesis, metabolism, and development. JAKs are multidomain proteins, in which the tyrosine kinase domain (JH1) is preceded by a pseudokinase domain (JH2). JH2 has critical regulatory functions and is a hotspot for many known oncogenic driver mutations. These mutations, which cause ligand-independent JAK activation, underlie various diseases—most notably haematopoietic malignancies caused by somatic JAK2 JH2 mutations. In the work presented here, we analysed the functions of JH2 and its mutations in the regulation of JAK activity. We found that all JAK JH2s have functional nucleotide-binding sites accessible to ATP and small molecule inhibitors. Most importantly, we found that disruption of ATP binding to JAK2 JH2 suppresses ligand-independent activation, thus identifying the JAK2 JH2 ATP-binding site as a potential drug target for the development of mutation-specific inhibitors. These inhibitors would be a distinct improvement over inhibitors of JAK2 currently used to treat MPNs, as current inhibitors do not distinguish between mutated and wild-type JAK2, and are unable to eradicate the disease. We also present a collaboration effort leading to a simulation-based model for JH2-mediated inhibition of JH1, thereby providing rationale for most known clinical JAK2 mutations. Moreover, we refine our understanding of ligand-mediated and ligand-independent activation of JAKs by presenting a systematic analysis of JAK2 mutations capable of inhibiting ligand-independent hyperactivation. We further identify a novel interface in JAK2 JH2, which is needed for heteromeric JAK2 activation in interferon-γ signalling

Nucleotide-binding mechanisms in pseudokinases

Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed

Connection between absorption properties and conformational changes in Deinococcus radiodurans phytochrome

Phytochromes consist of several protein domains and a linear tetrapyrrole molecule, which interact as a red-light-sensing system. In this study, size-exclusion chromatography and light-scattering techniques are combined with UV-vis spectroscopy to investigate light-induced changes in dimeric Deinococcus radiodurans bacterial phytochrome (DrBphP) and its subdomains. The photosensory unit (DrCBD-PHY) shows an unusually stable Pfr state with minimal dark reversion, whereas the histidine kinase (HK) domain facilitates dark reversion to the resting state. Size-exclusion chromatography reveals that all phytochrome fragments remain as dimers in the illuminated state and dark state. Still, the elution profiles of all phytochrome fragments differ between the illuminated and dark states. The differences are observed reliably only when the whole UV-vis spectrum is characterized along the elution profile and show more Pfr-state characteristics at later elution volumes in DrBphP and DrCBD-PHY fragments. This implies that the PHY domain has an important role in amplifying and relaying light-induced conformational changes to the HK domain. In the illuminated state, the HK domain appears partially unfolded and prone to form oligomers. The oligomerization of DrBphP can be diminished by converting the molecule back to the resting Pr state by using far-red light

Control of protein stability by post-translational modifications

Here the authors summarize current knowledge of the regulation of protein stability by various post-translational modifications (PTMs) including methylation and phosphorylation. PTM-regulated degrons act as signals for protein degradation or stabilization

Glycolytic flux-signaling controls mouse embryo mesoderm development.

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here, we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization