Carol V Robinson

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. Here, we identify Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Cells lacking Dmt1 exhibit defects in Notch signaling, accompanied by disruptions in autophagosomal and endolysosomal functions. Dmt1 deficiency leads to elevated Fe2+ levels, disturbed ferritin dynamics, heightened cytoplasmic and mitochondrial reactive oxygen species (ROS), increased lipid peroxidation, and altered autophagic flux. Consequently, these changes culminate in reduced lysosomal activity, impeding the degradation of Lamp1 and cleaved Notch1 (NICD1) proteins. Notably, Dmt1 exists in two isoforms, with and without an iron response element (ire). Our investigation reveals that silencing Dmt1-ire …

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores upon cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Surprisingly, cleavage-deficient D275A GSDMD is also palmitoylated after inflammasome stimulation or treatment with ROS activators, and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage, and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron …

CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O [C@ H] 1 [C@ H](O)[C@@ H](CO) O [C@@] 1 (CO) O [C@@ H] 1 [C@ H](O)[C@@ H](O)[C@ H](O)[C@@ H](CO) O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 10ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2, 3-di (octadeca-9, 12-dienoyloxy) propoxy-oxidophosphoryl] oxy-2-hydroxypropyl] 2, 3-di (octadeca-9, 12-dienoyloxy) propyl phosphate Chemical compound [Na+].[Na+]. CCCCCC= CCC= CCCCCCCCC (= O) OCC (OC (= O) CCCCCCCC= CCC= CCCCCC) COP ([O-])(= O) OCC (O) COP ([O-])(= O) OCC (OC (= O) CCCCCCCC= CCC= CCCCCC) COC (= O) CCCCCCCC= CCC= CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 10

The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane …

The peptidoglycan pathway represents one of the most successful drug targets with the last but critical step being the recycling of undecaprenyl phosphate. This translocation of undecaprenyl phosphate is facilitated by DedA and DUF386 domain-containing family membrane proteins via unknown mechanisms. Here we employ native mass spectrometry to investigate the interactions of Bacillus subtilis UptA, a putative C55-P flippase, with C55-P, membrane phospholipids and cell wall targeting antibiotics. Our results show that UptA, expressed and purified in E. coli, forms monomer-dimer equilibria, and binds to ligands in a pH-dependent fashion. Specifically, we show that UptA interacts more favourably with C55-P over shorter-chain analogues and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly intercept UptA function by out-competing the substrate for the protein binding site. Overall, this study illuminates the substrate-binding specificity of UptA, its potential regulation by anionic phospholipids, and provides insights for future development of antibiotics targeting carrier lipid recycling.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic–lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes …

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 μM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding …

The biomechanical properties and responses of tissues underpin a variety of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. Seemingly a multi-functional protein that interacts with a variety of partners, how FLNC is regulated at the molecular level is not well understood. Here we have investigated its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We found that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we have solved by means of X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work rationalises on the molecular level how FLNC might switch between stabilising functions in the cell, and reveals how HSPB7 acts as a specific molecular chaperone that regulates FLNC.