Biology subjects

Airola, M. V.

Publications and source records attributed to Airola, M. V..

12 recordsLinked to original sources

bioRxiv2026

Dithionite quenching of NBD-labeled lipids reveals artificial lipid droplet purity and neutral lipid surface accessibility

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionites ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied dithionite quenching to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

biochemistry↗

bioRxiv2026

Nir1-Nir2 Heterodimerization Confers Robustness to the Phosphoinositide Cycle

The phosphatidylinositol (PI) cycle sustains phosphoinositide-calcium signaling by replenishing plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) consumed during receptor activation. Recruitment of the lipid transfer protein Nir2 to endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (also known as ER-PM junctions) is central to this process, enabling transfer of PI from the ER to the PM for PIP2 resynthesis. Although the disease-associated Nir2 paralog Nir1 is essential for Nir2 recruitment under physiological conditions, the molecular basis of this regulation has remained unresolved. Here, we identify a conserved Nir Dimerization (NirD) domain in both Nir1 and Nir2 and determine the crystal structures. We show that NirD mediates preferential Nir1-Nir2 heterodimerization, which promotes Nir2 recruitment to ER-PM junctions and enhances PIP2 replenishment in stimulated cells. Furthermore, Nir1-Nir2 heterodimerization confers stimulus-strength-dependent, graded recruitment of Nir2, thereby broadening the sensitivity and dynamic range of PI cycle activity. Together, our findings uncover the structural mechanism underlying Nir1-dependent regulation of Nir2 and reveal paralog heterodimerization as a key strategy for scaling lipid transport at membrane contact sites to signaling demand, ensuring robust phosphoinositide homeostasis.

cell biology↗

bioRxiv2026

Van der Waals interactions mediate the enantiomeric substrate preference of the CTP:phosphoglycerate cytidylyltransferase CpgD

Caulobacter crescentus is a gram-negative bacterium that produces the anionic sphingolipid ceramide diphosphoglycerate that can substitute for the lipopolysaccharide component of the outer membrane. ccna_01210 is a gene in the operon for ceramide diphosphoglycerate synthesis and encodes for the enzyme, CpgD. CpgD is a magnesium-dependent CTP:phosphoglycerate cytidylyltransferase that catalyzes the synthesis of CDP-glycerate, and a pyrophosphate byproduct. CpgD displays substrate specificity for the nucleotide CTP and a preference for 2-phospho-D-glycerate over other phosphoglycerate enantiomers and isomers. Here, we present five high resolution structures for CpgD in various catalytic states that rationalize the specificity and preference of CpgD for its two substrates. This includes structures of apo CpgD, a CpgD-CTP-Mg2+ ternary complex, and three CpgD-CDP-glycerate-pyrophosphate-Mg2+ product bound complexes. The structures reveal CpgD nucleotide specificity is mediated by favorable hydrogen bonding interactions with the cytosine nucleobase of CTP, while the preference for 2-phospho-D-glycerate occurs due to favorable van der Waals interactions with the 2D enantiomer and unfavorable steric clashes with the 2L enantiomer. A catalytic mechanism involving a pentacoordinate transition state is proposed based on the observed stereochemical inversion of the -phosphate in the substrate CTP in comparison to the -phosphate of the product CDP-glycerate. Overall, this provides insights into the catalytic mechanism, nucleotide specificity, and enantiomeric substrate preference of the cytidylyltransferase CpgD that participates in a unique pathway of bacterial sphingolipid synthesis.

biochemistry↗

bioRxiv2026

Neutral Sphingomyelinase-2 Restrains TAZ to Suppress Breast Tumor Growth

Loss of tumor suppressor gene (TSG) activity is pervasive across cancers and linked to worse clinical outcomes, yet therapeutic efforts aimed at restoring TSGs have remained elusive. One underexplored avenue to address this problem is the targeting of metabolic signaling pathways that actively enforce tumor suppressive programs. Ceramide (Cer), the central hub of the sphingolipid (SL) metabolic network, has long been thought to have tumor suppressive functions, though its mechanistic roles remain incompletely defined. Here, we identify neutral sphingomyelinase-2 (nSMase2) as a critical mediator of Cer-dependent tumor suppression. We show that nSMase2 is frequently suppressed in breast cancer (BC) and its restoration inhibits tumorigenesis. Biologically, this was linked to the suppression of anchorage-independent growth (AIG) and to restraint of the HIPPO pathway effector TAZ, but not its paralog YAP. Taken together, these findings define a previously unrecognized metabolic tumor suppressor pathway, clarify ambiguities in both SL and HIPPO signaling networks, and highlight reactivation of nSMase2-Cer signaling as a potential therapeutic strategy in BC.

cancer biology↗

bioRxiv2025

DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains

Hereditary spastic paraplegia subtype SPG54 is a genetic neurological disorder caused by mutations in the DDHD2 gene. Excessive lipid droplet accumulation is observed in the brains of SPG54 patients and DDHD2 knockout mice, consistent with DDHD2s reported neutral lipase activity. Here, we find recombinant human DDHD2 preferentially hydrolyzes diacylglycerol (DAG) over phospholipids, with a slight preference for DAG over triacylglycerol (TAG). DDHD2 also exhibits transacylase activity, which enables transfer of acyl chains from triacylglycerols to diacylglycerols and monoacylglycerols to remodel the acyl chains of triglycerides. A predicted hydrophobic amphipathic helix on DDHD2 is essential for lipid droplet binding in vitro and in cells, and its lack reduces the enzymatic activity and triglyceride acyl chain remodeling. Adipose triglyceride lipase (ATGL), but not hormone sensitive lipase (HSL), also has transacylation activity and can remodel triglyceride acyl chains, but to a lesser extent than DDHD2. Taken together, this provides evidence that DDHD2 is a neutral lipid lipase and transacylase whose broad specificity enables triglyceride acyl-chain remodeling. SIGNIFICANCE STATEMENTTriglycerides (TAGs), the primary form of long-term energy storage, have acyl chain compositions crucial for diverse cellular processes. Lipases typically hydrolyze TAGs into free fatty acids. Here, we reveal a novel function for the neutral lipid lipase DDHD2: a transacylase activity. Instead of releasing fatty acids, DDHD2 transfers them between neutral lipids, altering TAG acyl chain composition. This transacylation requires the unique oil environment of lipid droplets (LDs), which excludes water from DDHD2s lipolytic active site, favoring transacylation over hydrolysis. DDHD2s lipase and transacylase activities enable TAG acyl-chain remodeling, demonstrating the possibility that a single enzyme can catalyze TAG cycling. This finding has implications for understanding lipid metabolism, LD dynamics, and specific motor neuron diseases implicating DDHD2.

biochemistry↗

bioRxiv2025

Structures of a lipin/Pah phosphatidic acid phosphatase in distinct catalytic states reveal a signature motif for substrate recognition

Lipin/Pah phosphatidic acid phosphatases (PAPs) are Mg2+-dependent enzymes that catalyze the dephosphorylation of phosphatidic acid (PA) to produce diacylglycerol. Deficiency of lipin PAP activity in humans results in inflammatory disorders such as rhabdomyolysis and Majeed syndrome. Previously, we reported the first PAP enzyme structures of Tetrahymena thermophila (Tt) Pah2 at 3.0[A] resolution. Here, we present five new higher resolution (1.95-2.40[A]) structures of Tt Pah2, in distinct catalytic states, which represent active states of catalysis, including a phospho-Asp transition state mimic, and an inactive state with a distorted active site. The structures, in conjunction with flexible docking simulations and biochemical analysis, connect two highly conserved aspartate and arginine residues in magnesium coordination and recognition of the substrate PA. Overall, this provides a structural basis for catalysis and defines a signature Asp-Arg motif in lipin/Pah PAPs that enables recognition of their lipid substrate PA, providing insight into how the HAD domain of lipin/Pah PAPs evolved to act on a membrane embedded substrate.

biochemistry↗

bioRxiv2024

Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production

Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analyzing the predicted structure of the LNS2 domain from the lipid transfer protein Nir1, we suspected that this phosphatidic acid-interacting Lipin-like sequence of Nir1 (PILS-Nir1) could serve as a novel PA biosensor. We then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells expressing a fluorescent PILS-Nir1 to determine how specific lipids affect the interaction of PILS-Nir1 with membranes. We found that PILS-Nir1 bound to both PA and PIP2 in vitro. However, only PA was necessary and sufficient to localize PILS-Nir1 to membranes in cells. PILS-Nir1 also showed a heightened responsiveness to PA produced in various organelles when compared to biosensors using the Spo20 PA binding domain. PILS-Nir1s high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, PILS-Nir1 emerges as a versatile and sensitive biosensor, offering a new powerful tool for real-time investigation of PA dynamics in live cells.

cell biology↗

bioRxiv2023

Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a non-canonical protein serine/threonine phosphatase that regulates ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of CTDNEP1 or NEP1R1 in human cells generate identical phenotypes, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a pseudo-substrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

biochemistry↗

bioRxiv2023

Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage

The endoplasmic reticulum (ER) is the site for the synthesis of the major membrane and storage lipids. Lipin 1 produces diacylglycerol, the lipid intermediate critical for the synthesis of both membrane and storage lipids in the ER. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but its role in lipid storage in mammalian cells is unknown. Here, we show that the ubiquitin-proteasome degradation pathway controls the levels of ER/nuclear envelope-associated CTDNEP1 to regulate ER membrane synthesis through lipin 1. The N-terminus of CTDNEP1 is an amphipathic helix that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 with its regulatory subunit NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate a role for NEP1R1 in temporarily shielding CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, we found that NEP1R1 is not required for CTDNEP1s role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on its regulatory subunit differs during ER membrane synthesis and lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis and storage under fluctuating conditions.

cell biology↗

bioRxiv2022

Structure and dynamics of human perilipin 3 membrane association

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyzed how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We found the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG enriched membranes. Membrane binding induces a disorder/order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

biochemistry↗

bioRxiv2021

The middle lipin (M-Lip) domain is a new dimeric protein fold that binds membranes

Phospholipid synthesis and fat storage as triglycerides is regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized and novel protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in full-length lipin 1 influences PAP activity, membrane binding, subcellular localization, oligomerization, and adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a new dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

biochemistry↗

bioRxiv2018

Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+ dependent amidases

Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are the members of the CREST superfamily of integral-membrane lipid hydrolases, including the adiponectin receptors which play roles in energy metabolism. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. However, the structural and catalytic roles for these residues are unclear. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six point mutantions of the conserved CREST motif were developed that are predicted to form a Zn-dependent active site based on homology with the human adiponectin receptors, whose crystal structures were recently determined. Five mutations completely lost their activity, except for S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutation was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state and differs from the proposed role in Zinc coordination for the adiponectin receptors (Vasiliauskaite-Brooks et. al., Nature, 2017). Together, these data suggest ACER3 is a Zn2+-dependent amidase that uses a catalytic mechanism for ceramide hydrolysis that is similar to other soluble Zn-based amidases. Consistent with this mechanism, ACER3 was specifically inhibited by trichostatin A, an HDAC inhibitor, which is a strong chelator of Zinc.

biochemistry↗