Biology subjects

Januliene, D.

Publications and source records attributed to Januliene, D..

13 recordsLinked to original sources

bioRxiv2026

Cholesterol transport cycle of human ABCA2

Cholesterol is a key component of cellular membranes and is critical for brain function, particularly axon myelination. Among the 48 human ATP-binding cassette (ABC) transporters, ABCA2 exhibits the highest expression in the brain and is involved in cholesterol metabolism, primarily in oligodendrocytes. Notably, ABCA2 has been associated with myelin sheath integrity and maintenance, as well as Alzheimer's disease. Here, we report cryo-EM structures of human ABCA2 that reveal critical endogenous lipid-binding sites unique to ABCA2. Our five distinct conformations include a previously uncharacterized intermediate between the closed and apo states of ABCA subfamily transporters. Most importantly, we elucidated the cholesterol transport mechanism of ABCA2, which involves novel interdependent rotations of the exocytoplasmic domains (ECDs) and regulatory domains (RDs). Our structural findings provide a new perspective on ABCA transporter function and highlight the role of ABCA2 in facilitating efficient cholesterol recycling and transport in the brain.

molecular biology↗

bioRxiv2026

A dual sensor regulates P-glycoproteins structural plasticity

P-glycoprotein is an efflux pump with an exceptionally broad substrate profile which drives its profound clinical impact. Despite its biological importance, it remains obscure how P-glycoprotein achieves its polyspecificity and how substrate binding and the lipid environment stimulate its activity. Structural data highlight the importance of transmembrane helices 4 and 10, which surround the binding pocket, and identify them as key players in substrate recognition. Here, we used cryo-EM to study P-glycoprotein in detergent and nanodiscs to strategically leverage environment- and substrate-dependent phenotypes. This approach allowed us to decipher unexpected and distinct roles of transmembrane helices 4 and 10, which structurally explain differences in ATPase activity. Our data highlights helix 4 as an environment sensor and helix 10 as the key player in substrate recognition constituting a dual regulation mechanism for the functional plasticity of P-glycoprotein, and visualizes the intricate interplay between a membrane protein and its environment.

molecular biology↗

bioRxiv2025

Soluble SORL1 in cerebrospinal fluid as a marker for functional impact of rare SORL1 variants.

BackgroundAs part of the retromer, sortilin-related receptor (SORL1) sorts cargo proteins away from the endosome to the trans-Golgi network or to the cell surface, where SORL1 is cleaved into sSORL1 and shed into the interstitial fluid and CSF. SORL1s cargo includes APP and A{beta}, which may explain why protein-truncating genetic variants (PTVs) in SORL1 are observed almost exclusively in Alzheimers Disease (AD) patients, and that rare, predicted pathogenic missense variants have been associated with a 10-fold increased risk of AD. However, functional evidence supporting variant pathogenicity is warranted. Here we investigated whether soluble SORL1 (sSORL1) concentrations in cerebrospinal fluid (CSF) offer a potential in vivo biomarker to support impaired SORL1 function in variant carriers. MethodsUsing an ELISA assay for SORL1 (ABCAM), we determined sSORL1 concentrations in CSF from 218 participants of the Alzheimer Dementia Cohort (ADC) (54% females). We compared sSORL1 in CSF derived from 90 carriers of diverse SORL1 variants with concentrations observed in 78 SORL1-WT AD patients, and 50 SORL1-WT controls for whom CSF-pTau-181, CSF-tTau, CSF-A{beta}42 concentrations were available. In a subset of 36 individuals, we used Western blotting (WB) to validate sSORL1 concentrations as determined by ELISA. ResultsCSF-sSORL1 concentrations did not differ between SORL1-WT AD patients and controls. While CSF-sSORL1 did not correlate with CSF-A{beta}42 concentrations in SORL1-WT AD patients (p=0.62), it correlated with sCSF-ptau-181 (p=9.7x10-6). The mean CSF-sSORL1 concentration in the SORL1 WT AD cases and controls was 466 pg/ml, with wide variance (SD = 133). Comparatively, concentrations were significantly lower in PTV carriers (260 pg/ml, p=3.2x10-7) and in carriers of predicted damaging SORL1 missense variants (323 pg/ml, p=2.4x10-7). CSF-sSORL1 concentrations measured by ELISA correlated strongly with concentrations estimated by WB ({rho}=0.552; p=5.0x10-4). ConclusionCSF-sSORL1 increases with CSF-ptau, suggesting that increased SORL1-retromer activity may serve to rescue cellular stress associated with AD-related processes. However, impairing SORL1 genetic variants may preclude SORL1 trafficking to the cell surface, as supported by lower CSF-sSORL1 protein concentrations in carriers. While further refinements are necessary, we present first evidence for the applicability of ELISA-based quantification of sSORL1 in CSF to evaluate the functional impact of rare SORL1 variants.

neuroscience↗

bioRxiv2025

Mechanistic Plasticity of the RabGEFs Mon1-Ccz1 and Fuzzy-Inturned

Rab GTPases organize intracellular trafficking and provide identity to organelles. Their spatiotemporal activation by guanine nucleotide exchange factors (GEFs) is tightly controlled to ensure fidelity. Our structural and functional comparison of the tri-longin domain RabGEFs Mon1-Ccz1 and Fuzzy-Inturned reveals the molecular basis for their target specificity. Both complexes rely on a conserved sequence motif of their substrate GTPases for the catalytic mechanism, while secondary interactions allow discrimination between targets. We also find that dimeric Mon1-Ccz1 from fungi and the metazoan homologs with the additional third subunit RMC1/Bulli bind membranes through electrostatic interactions via distinct interfaces. Protein-lipid interaction studies thus reveal a function of RMC1/Bulli in the complex as mediator of membrane recruitment. In the case of Fuzzy-Inturned, reconstitution experiments demonstrate that the BAR (Bin-Amphiphysin-Rvs) domain protein CiBAR1 can support membrane recruitment of the GEF. Collectively, our study demonstrates the molecular basis for the adaptation of TLD-RabGEFs to different cellular functions.

biochemistry↗

bioRxiv2024

The ABC transporter MsbA in a dozen environments

High-resolution structure determination of membrane proteins typically requires isolation from the native lipid bilayer and reconstitution into artificial membrane mimics. For this purpose, numerous detergents, amphipols, polymers and membrane scaffold proteins are available. The choice of the specific membrane substitute can strongly affect the proteins specific activity, stability and conformational spectrum, potentially leading to errors or misinterpretation during analysis. The bacterial ATP-binding cassette transporter MsbA is a prominent example of such environment-specific bias, resulting in apparent conformational and activity responses. Here, we present a systematic analysis of the conformational spectrum of MsbA, stabilized in a dozen environments, using cryo-EM. Our data show pronounced structural feedback of the ABC transporter to the respective membrane mimetics. Detergents generally favour a conformation with wide separation of the nucleotide-binding domains, while nanodiscs induce the narrow conformation. Notably, only three of the dozen tested environments allow MsbA to sample the functional conformational spectrum, enabling full movement of the nucleotide-binding domains between narrow and wide inward-facing conformations. We expect this study to serve as a blueprint for other membrane proteins, even where the structural reaction to the hydrophobic environment is not directly visible but still critical for the proteins function.

molecular biology↗

bioRxiv2024

The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.

The levels of sphingolipids are crucial determinants of neurodegenerative disorders. Therefore, sphingolipid levels have to be tightly regulated. The Orm protein family and ceramides act as inhibitors in the rate-limiting step of sphingolipid biosynthesis - the condensation of L-serine and palmitoyl-CoA. The two yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While the sequences of the Orm proteins are highly similar, however, Orm1 and Orm2 are differentially regulated in yeast cells. The mechanistic details of the differential regulation remain elusive. To elucidate the regulatory mechanism, we determined the cryo-electron microscopy structure of the SPT complex containing Orm2. Through in vitro activity assays and a newly developed plasmid coverage assay combined with targeted lipidomics, we demonstrate that the SPT complex with Orm2 exhibits lower activity in comparison to the complex containing Orm1. This collectively suggests a higher inhibitory potential of Orm2, despite the remarkably similar structures of the Orm1- and Orm2 containing complexes. The high conservation of the SPT from yeast to man implies that the three human ORMDL isoforms could also have different regulatory capacities, which might be essential to understanding their role in sphingolipid-mediated neurodegenerative disorders. HighlightsO_LICryo-EM structures of serine palmitoyltransferase with Orm2 from S. cerevisiae C_LIO_LICeramide-mediated inhibition on SPT activity is conserved amongst SPTs C_LIO_LIThe two yeast Orm isoforms have different regulatory capacity C_LI

biochemistry↗

bioRxiv2023

Structure of the yeast ceramide synthase

Ceramides play a pivotal role as essential lipids, serving as foundational components for complex sphingolipids and potent signaling molecules. Ceramides are the products of the N-acylation of a sphingoid base and a CoA-activated fatty acid. This reaction is catalyzed by the enzymes of the evolutionarily conserved ceramide synthase (CerS) family. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here, we employed cryo-EM single particle analysis to unravel the structure of the yeast ceramide synthase complex in both an active and a fumonisin B1 inhibited state. Our findings shed light on the complexs architecture, revealing a dimer of Lip1 subunits bound to the two catalytic subunits, Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice that is accessible from both the cytosolic site as well as from the intermembrane space of the endoplasmic reticulum (ER). Within this cavity, we identify amino acids forming the active center and a sphingoid base, one of the substrates of the complex. Together, this suggests a pre-loaded state of the CerS. Additionally, the fumonisin B1 bound structure reveals the inhibitory mechanism by blocking the cytosolic acyl-CoA binding site.

biochemistry↗

bioRxiv2023

Cryo-EM structure of cell-free synthesized human histamine H2 receptor coupled to heterotrimeric Gs protein in lipid nanodisc environment

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 [A]. The complex was generated by cotranslational insertion into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. It is the first structure obtained by this detergent-free strategy and the first GPCR/Gs complex structure in lipid environment. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

molecular biology↗

bioRxiv2023

Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyl-transferase (SPT) catalyzes the rate limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

biochemistry↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗

bioRxiv2022

Mechanism of multimodal substrate translocation in P-glycoprotein

P-glycoprotein (Pgp) is a prototypical ABC transporter of great biological and clinical significance that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs1-3. Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds4-9, but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.

biophysics↗

bioRxiv2020

SorCS1 binds the insulin receptor to enhance insulin sensitivity

Type 2 diabetes has reached endemic proportions and is a substantial burden for the affected patients and the society. Along with lifestyle factors, a number of genetic loci predisposing to type 2 diabetes have been identified, including SORCS1 that encodes the transmembrane receptor SorCS1. The ectodomain of SorCS1 (sol-SorCS1) is shed from plasma membranes but the biological function of this fragment is unknown. Here we show that sol-SorCS1 acts as a high-affinity binding partner for the insulin receptor to stabilize the receptor and increase insulin affinity, protein kinase B activation, and glucose uptake in myocytes. Sol-SorCS1 is liberated from adipocytes, and in diabetic patients the plasma concentration positively correlates with body mass index, but inversely with plasma glucose. In mouse models of insulin resistance, exogenous sol-SorCS1 restored insulin sensitivity. We conclude that sol-SorCS1 increases peripheral insulin sensitivity and propose sol-SorCS1 as a novel insulin sensitizing adipokine and potential antidiabetic agent.

physiology↗

bioRxiv2019

Structure and autoregulation of a P4-ATPase lipid flippase

P4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of the Saccharomyces cerevisiae Drs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

molecular biology↗