Synaptic transmission occurs synchronously with real-world events, far faster than vesicle fusion for hormone release or membrane biogenesis, all mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. How SNAREs cooperate to achieve synchronous neurotransmitter release is a long-standing mystery. Rapid release (<7 milliseconds) has been reconstituted from purified synaptic SNAREs, SNARE-assembling chaperones, and calcium ion sensors in a fully-defined, genetically validated system that enables single-molecule counting in docked vesicles before release. SNAREpin complexes (12 ± 0.3) are found in each such ready-release vesicle, suggesting a regular structure. Several genetic conditions (including point mutation of the synaptic vesicle protein Synaptophysin from a Synaptopathy patient and human and mouse disease mutations of the synaptic vesicle protein vesicle-associated membrane protein-2 (VAMP2) reduce the number of SNAREpins to 6 ± 0.3 and result in profoundly delayed release over 0.1 to 1 seconds. Omitting Synaptophysin, whose hexamers preassemble 12 copies of VAMP2, also yields ~6 SNAREpins and delays release.

Lipofuscin is an autofluorescent material that accrues in brain tissues with age and in Neuronal Ceroid Lipofuscinosis (NCL), a neurodegenerative disease with pediatric onset. The distribution, composition, and organellar origin of lipofuscin have remained unclear despite its widespread presence in aged tissues and involvement in neurodegeneration. Here, we elucidate lipofuscin composition in mouse and human brain and assemble a reference neuroanatomical atlas of lipofuscin accumulation with age and NCL (Type 1; CLN1) progression across 425 fine brain regions. We identify a primary role of the lysosomal-mitochondrial axis in the formation of lipofuscin pathology via multimodal mass spectrometry, ultrastructural analyses, and assays of cellular and enzymatic metabolism. We find the protein and lipid composition of lipofuscin in the aged and CLN1 brain to be remarkably similar. Dissection of implicated molecular pathways reveals protein S-acylation and unsaturated lipid homeostasis as central processes involved in lipofuscin deposition during aging and CLN1. Notably, > 95

Peripheral membrane proteins (PMPs) are critical mediators of signaling cascades initiated at the cell surface. Their functions depend on their innate ability to interact dynamically with membranes in response to changing cellular conditions. This membrane recruitment may occur via high-affinity interactions with specific lipids/proteins or via transient, low-affinity interactions with the membrane. These weak and dynamic interactions, which are critical regulators of PMP function, are challenging to capture. Taking Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell activation, we demonstrate a native mass spectrometry platform to understand lipid-mediated recruitment of PMPs by directly studying it from lipid bilayers customized to target membranes. Our data demonstrate that BTK recognizes phosphatidylserine (PS) independently of phosphatidylinositol (3, 4, 5) phosphate (PIP3) binding. We show that PS-bound BTK retains PIP3 binding via high-affinity sites, while exhibiting PIP3-independent basal membrane recruitment. Biochemical assays show that this PS-mediated recruitment sensitizes BTK to PIP3-mediated activation at near-physiological PIP3 concentrations. Thus, we propose a two-step model for BTK membrane recruitment and activation. A low-affinity interaction with high-copy number PS enables plasma membrane recruitment of BTK and increases its membrane-bound concentration. Upon B cell activation, this prerecruited, membrane-bound BTK population localizes to PIP3-rich domains via electrostatic gliding along the membrane, driven by low-affinity PS and high-affinity PIP3 binding. This indicates a cooperative mechanism in which PS can amplify B cell signaling by increasing membrane-bound BTK levels. Our work demonstrates a general model of regulation of PH domain-containing proteins by weak protein-lipid interactions, which can be extended to other PMPs.

Lipofuscin is an autofluorescent material that accrues in brain tissues with age and in Neuronal Ceroid Lipofuscinosis (NCL), a neurodegenerative disease with pediatric onset. The distribution, composition, and organellar origin of lipofuscin have remained unclear despite its widespread presence in aged tissues and involvement in neurodegeneration. Here, we elucidate lipofuscin composition in mouse and human brain and report the spatiotemporal dynamics of lipofuscin accumulation in aging and NCL in a murine neuroanatomical atlas. Multimodal mass spectrometry, ultrastructural analyses, and assays of metabolic flux identify a primary role of the lysosomal-mitochondrial axis in the formation of lipofuscin pathology. Dissection of implicated molecular pathways reveals protein S-acylation and unsaturated lipid homeostasis as central processes involved in lipofuscin deposition during aging and NCL.

Macromolecular organization of proteins and lipids in cellular membranes is fundamental to cell functionality. Recent advances in native mass spectrometry (nMS) have established it as a key analytical tool for capturing these associations. This typically necessitates the extraction of target membrane proteins (MPs) from their physiological environments into detergent-like surroundings. In our recent studies using in vitro synthetic liposomes, we discovered that gas phase supercharging can selectively destabilize lipid bilayers and enable MS1 detection of embedded and associated protein-lipid complexes. Here, we further extend and apply this methodology to native cell-derived membrane vesicles. We demonstrate our ability to detect and ID protein complexes and their proteoforms directly from native membranes using supercharger-assisted prequadrupole activation followed by downstream native top-down tandem mass spectrometry, which combines both collision-based and electron capture-based fragmentation approaches. We first demonstrated this approach through native top-down identification of several integral MPs from in vitro membranes. Subsequently, we developed a protocol to produce nMS-ready native membrane vesicles. Applying to Escherichia coli total membranes, we generated nMS-ready vesicles and identified both integral and membrane-associated protein complexes of homomeric and heteromeric nature using our supercharging-enabled native top-down platform. For the heteropentameric β-barrel-assembly machinery (BAM) complex, which includes the integral MP BAM-A, we detected several lipidated proteoforms. For peripheral homodimeric dihydrolipoyl dehydrogenase, we identified bound endogenous metabolite cofactors. Furthermore, using BAM complex, a crucial antibiotic target, we show how this platform could be utilized to study drug binding to MPs directly from their native membranes.

Proper fuelling of the brain is critical to sustain cognitive function, but the role of fatty acid (FA) combustion in this process has been elusive. Here we show that acute block of a neuron-specific triglyceride lipase, DDHD2 (a genetic driver of complex hereditary spastic paraplegia), or of the mitochondrial lipid transporter CPT1 leads to rapid onset of torpor in adult male mice. These data indicate that in vivo neurons are probably constantly fluxing FAs derived from lipid droplets (LDs) through β-oxidation to support neuronal bioenergetics. We show that in dissociated neurons, electrical silencing or blocking of DDHD2 leads to accumulation of neuronal LDs, including at nerve terminals, and that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion to drive local mitochondrial ATP production. These data demonstrate that nerve terminals can make use of LDs during electrical activity to provide metabolic support and probably have a critical role in supporting neuron function in vivo.

Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create a negative electrostatic surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.

Lipofuscin is an autofluorescent material that accrues in brain tissues with age and in Neuronal Ceroid Lipofuscinosis (NCL), a neurodegenerative disease with pediatric onset. The distribution, composition, and organellar origin of lipofuscin have remained unclear despite its widespread presence in aged tissues and involvement in neurodegeneration. Here, we elucidate lipofuscin composition and report the spatiotemporal dynamics of lipofuscin accumulation in aging and NCL on a neuroanatomical atlas. Multimodal mass spectrometry, ultrastructural analyses, and assays of metabolic flux identify a primary role of the lysosomal-mitochondrial axis in lipofuscin formation. Dissection of implicated molecular pathways reveals protein S-acylation and lipid homeostasis as central processes involved in aging and NCL.

Background and Significance: Induction of ferroptosis, a form of cell death driven by iron-dependent peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids (PUFA-PL), has shown potential in therapy-resistant tumors. However, the factors determining sensitivity of cancer cells to this type of death are not well understood, and no potent ferroptosis inducers are available clinically. Our comparative analysis of publicly availablegene dependency and drug sensitivity data (lymphoblasts.org) identified B-cell lymphoma as one of the most ferroptosis-sensitive tumor types. We uncovered that B-cell lymphomas are highly enriched in PUFA and selectively dependent on pro-ferroptotic PUFA metabolism to maintain competitive fitness and lipid membrane properties, which endows them with an intrinsic vulnerability to ferroptotic cell death. Results: Our comparative analyses of public drug screening data (CTD, GDSC) and a validation screen we performed uncovered that B-cell lymphomas are the most sensitive type of tumor to all evaluated ferroptosis inducers, including GPX4 inhibitors (RSL3, ML210), the inhibitor of the cystine/glutamate antiporter system Xc- erastin, and the iron oxidizer FINO2. To further understand the factors contributing to this ferroptosis vulnerability, we performed comparative analyses of CRISPR dependency screens from the Cancer Dependency map. Unexpectedly, this approach uncovered ACSL4, a key enzyme in PUFA-PL production, as a selective B-cell dependency. This was unexpected because ACSL4 induces sensitivity to ferroptosis through production of long-chain PUFA-PL, especially containing arachidonic acid. In line with this surprising dependency, comparative analysis of Cancer Cell Line Encyclopedia metabolomics data showed a marked enrichment in multiple types of PUFA in B-cell lymphomas. Similarly, data from the Immunological Proteomic Resource showed that ACSL4 is one of the most upregulated proteins upon B-cell activation, suggesting a key role of PUFA metabolism during increased energetic demands. Furthermore, high expression of ACSL4 was associated with decreased overall survival in the MMMLNP diffuse large B-cell lymphoma clinical trial cohort, while the opposite trend was observed with expression of ACSL3, which counteracts PUFA-PL by driving metabolism of anti-ferroptotic monounsaturated fatty acids. A PUFA-rich gene expression signature strongly predicted prognosis, as indicated by a hazard ratio for mortality of 2.484 in ACSL4high/ACSL3low individuals (95% confidence interval: 1.709 – 3.609; measured by log-rank test). In line with the key role of PUFA in ferroptosis regulation, our whole-genome CRISPR knockout screen performed under the selective pressure of RSL3 showed that ACSL4 is one of the key genes promoting ferroptosis sensitivity in B-cell malignancies, while the opposite was seen with ACSL3. These findings suggest that, while B-cell lymphomas are dependent on PUFA metabolism, this dependence might represent a vulnerability making them highly sensitive to ferroptosis. To evaluate the roles of ACSL4 and PUFA-PL in B-cell lymphomas, we utilized CRISPR-mediated homology-directed repair to knock-in an ACSL4 degradation tag (dTAG) in B-cell lymphoma cell lines. dTAG induction led to complete loss of ACSL4 within one hour and led to progressive increase in resistance to lipid peroxidation, consistent with loss of PUFA-PL at cell membranes. While loss of ACSL4 promoted ferroptosis resistance, it also led to cell depletion in competitive growth assays, indicating loss of competitive fitness upon PUFA-PL depletion. Molecular biophysics experiments showed that ACSL4 loss and resulting PUFA-PL depletion increases membrane flow resistance, as measured by an increase in membrane cytoskeleton attachment (9.246 ± 1.43 x 105 vs 5.141 ± 1.57 x 105 pN3s/μM for ACSL4 loss vs negative control, p = 0.00015). PUFA-PL loss thus compromises membrane properties of B-cell lymphomas, which might affect key membrane-dependent functions. Conclusions: We show that B-cell lymphomas are selectively dependent on PUFA metabolism to maintain membrane properties and competitive fitness. However, this intrinsic metabolic dependency is a key factor making them highly vulnerable to ferroptosis. Our findings also provide insight into B-cell lymphoma metabolism and lipid membrane dynamics, and how it could be leveraged as a therapeutic strategy to potently induce ferroptosis in B-cell lymphomas.

Microbial extracellular electron transfer (EET) drives various globally important environmental phenomena and has biotechnology applications. Diverse prokaryotes have been proposed to perform EET via surface-displayed “nanowires” composed of multi-heme cytochromes. However, the mechanism that enables only a few cytochromes to polymerize into nanowires is unclear. Here, we identify a highly conserved omcS-companion (osc) cluster that drives the formation of cytochrome OmcS nanowires in Geobacter sulfurreducens. Through a combination of genetic, biochemical, and biophysical methods, we establish that prolyl isomerase-containing chaperon OscH, channel-like OscEFG, and β-propeller-like OscD are involved in the folding, secretion, and morphology maintenance of OmcS nanowires, respectively. OscH and OscG can interact with OmcS. Furthermore, overexpression of oscG accelerates EET by overproducing nanowires in an ATP-dependent manner. Heme loading splits OscD; ΔoscD accelerates cell growth, bundles nanowires into cables. Our findings establish the mechanism and prevalence of a specialized and modular assembly system for nanowires across phylogenetically diverse species and environments

This is a protocol to conduct Native Nanobleach of native nanodiscs.

This is a protocol for using the extraction database found at www.polymerscreen.yale.edu for optimal extraction of membrane proteins into native nanodiscs.

Cellular membrane plays an essential role in regulating hierarchical organizations of membrane proteins (MP) and lipids that drive downstream signaling cascades. While native mass spectrometry (nMS) has been at the forefront of detecting these complexes, studying MPs with nMS demands prior dissolution of the cellular membrane, such as using detergents. This critical prerequisite to dissolve the membrane prior to MS-analysis often leads to the loss of critical protein-protein/lipid interactions, severely limiting applicability of the approach in studying membrane biology.

The protocol gives details for synthesizing the ChloroSMA series of membrane active polymers.

Transient association of peripheral membrane proteins (PMPs) with cellular membranes is vital to regulating a myriad of signaling pathways central to human health and disease. Increasing evidence suggests lipids play a pivotal role in regulating membrane recruitment, oligomerization, and activation of PMPs. Nevertheless, it remains experimentally challenging to detect these interactions and their effects directly from a native-like membrane environment. We have recently established a native mass spectrometry (nMS) method to study integral membrane proteins from tunable lipid bilayers and investigate membrane-associated oligomeric states and lipid interactors (Panda et.al.