Simulation study of membrane bending by protein crowding: a case study with the epsin N-terminal homology domain.

The mechanisms by which peripheral membrane proteins generate curvature is currently an active area of research. One of the proposed mechanisms is amphipathic insertion or the 'wedge' mechanism in which the protein shallowly inserts an amphipathic helix inside the membrane to drive the curvature. However, recent experimental studies have challenged the efficiency of the 'wedge' mechanism as it requires unusual protein densities. These studies proposed an alternative mechanism, namely 'protein-crowding', in which the lateral pressure generated by the random collisions among the membrane bound proteins drives the bending. In this study, we employ atomistic and coarse-grained molecular dynamics simulations to investigate the effects of amphipathic insertion and protein crowding on the membrane surface. Considering epsin N-terminal homology (ENTH) domain as a model protein, we show that amphipathic insertion is not essential for membrane bending. Our results suggest that ENTH domains can aggregate on the membrane surface by employing another structured region (H3 helix). And this protein crowding decreases the cohesive energy of the lipid tails which causes a significant decrease in the membrane bending rigidity. The ENTH domain can generate a similar degree of membrane curvature irrespective of the activity of its H0 helix. Our results are consistent with the recent experimental results.

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Arabidopsis clathrin adaptor EPSIN1 but not MODIFIED TRANSPORT TO THE VACOULE1 contributes to effective plant immunity against pathogenic Pseudomonas bacteria.

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin-1C-Terminal Domain.

Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.1.

Complexin Membrane Interactions: Implications for Synapse Evolution and Function.

The molecules and mechanisms behind chemical synaptic transmission have been explored for decades. For several of the core proteins involved in synaptic vesicle fusion, we now have a reasonably detailed grasp of their biochemical, structural, and functional properties. Complexin is one of the key synaptic proteins for which a simple mechanistic understanding is still lacking. Living up to its name, this small protein has been associated with a variety of roles differing between synapses and between species, but little consensus has been reached on its fundamental modes of action. Much attention has been paid to its deeply conserved SNARE-binding properties, while membrane-binding features of complexin and their functional significance have yet to be explored to the same degree. In this review, we summarize the known membrane interactions of the complexin C-terminal domain and their potential relevance to its function, synaptic localization, and evolutionary history.

A genome-wide search of Toll/Interleukin-1 receptor (TIR) domain-containing adapter molecule (TICAM) and their evolutionary divergence from other TIR domain containing proteins.

Toll/Interleukin-1 receptor (TIR) domains are cytoplasmic domain that mediates receptor signalling. These domains are present in proteins like Toll-like receptors (TLR), its signaling adaptors and Interleukins, that form a major part of the immune system. These TIR domain containing signaling adaptors binds to the TLRs and interacts with their TIR domains for downstream signaling. We have examined the evolutionary divergence across the tree of life of two of these TIR domain containing adaptor molecules (TICAM) i.e., TIR domain-containing adapter-inducing interferon-ß (TRIF/TICAM1) and TIR domain containing adaptor molecule2 (TRAM/TICAM2), by using computational approaches. We studied their orthologs, domain architecture, conserved motifs, and amino acid variations. Our study also adds a timeframe to infer the duplication of TICAM protein from Leptocardii and later divergence into TICAM1/TRIF and TICAM2/TRAM. More evidence of TRIF proteins was seen, but the absence of conserved co-existing domains such as TRIF-NTD, TIR, and RHIM domains in distant relatives hints on diversification and adaptation to different biological functions. TRAM was lost in Actinopteri and has conserved domain architecture of TIR across species except in Aves. An additional isoform of TRAM, TAG (TRAM adaptor with the GOLD domain), could be identified in species in the Mesozoic era. Finally, the Hypothesis based Likelihood ratio test was applied to look for selection pressure amongst orthologues of TRIF and TRAM to search for positively selected sites. These residues were mostly seen in the non-structural region of the proteins. Overall, this study unravels evolutionary information on the adaptors TRAM and TRIF and how well they had duplicated to perform diverse functions by changes in their domain architecture across lineages.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.

Structure of dynein-dynactin on microtubules shows tandem adaptor binding.

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4-6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9-13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

Molecular determinants of complexin clamping and activation function.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.

The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

ITSN1: a novel candidate gene involved in autosomal dominant neurodevelopmental disorder spectrum.

ITSN1 plays an important role in brain development. Recent studies in large cohorts of subjects with neurodevelopmental disorders have identified de novo variants in ITSN1 gene thereby suggesting that this gene is involved in the development of such disorders. The aim of this study is to provide further proof of such a link. We performed trio exome sequencing in a patient presenting autism, intellectual disability, and severe behavioral difficulties. Additional affected patients with a neurodevelopmental disorder harboring a heterozygous variant in ITSN1 (NM_003024.2) were collected through a worldwide collaboration. All patients underwent detailed phenotypic and genetic assessment and data was collected and shared by healthcare givers. We identified ten novel patients from eight families with heterozygous truncating or missense variants in ITSN1 gene. In addition, four previously published patients from large meta-analysis studies were included. In total, 7/14 patients presented a de novo variant in ITSN1. All patients showed neurodevelopmental disorders from autism spectrum disorders (90%), intellectual disability (86%), and epilepsy (30%). We demonstrated that truncating variants are in the first half of ITSN1 whereas missense variants are clustered in C-terminal region. We suggest ITSN1 gene is involved in development of an autism spectrum disorder with variable additional neurodevelopmental deficiency, thus confirming the hypothesis that ITSN1 is important for brain development.

Endocytic protein intersectin1-S shuttles into nucleus to suppress the DNA replication in breast cancer.

Breast cancer is the most common type of cancer worldwide. However, the well-known molecular biomarkers are not enough to meet the needs of precision medicine. In search for novel targets in this regard, we reported ITSN1 (intersectin1) as one of the candidates through mRNA microarray analysis. In the present study, we reported that endocytic protein ITSN1-S exists not only in the cytoplasm but also in nuclei of breast cancer cells. ITSN1-S' functional nuclear localization signal is within its residues 306-312. Its nuclear export signal (NES) resides within its SH3 domains. We also found, the interaction between the CC domain of nuclear ITSN1-S and the NT domain of nuclear DNA helicase II (NDH II) directly suppressed the DNA replication and nascent DNA synthesis by inhibiting the R-loops resolution in breast cancer cells. Furthermore, the interaction between the EH domains of cytoplasmic ITSN1-S and PI3KC2α inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway. Our results were confirmed in both ITSN1 gene knockout cells and in vivo assays. Finally, our clinical data showed a potential application of the combined consideration of the cytoplasmic and nuclear ITSN1-S as an independent prognosis factor. In conclusion, our study revealed ITSN1-S' novel positioning in the nuclei of breast cancer cells, its function in suppressing DNA replication, and its potential application in improved breast cancer prognosis.

Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.

Detailed structure of mouse interferon α2 and its interaction with Sortilin.

Interferon α (IFNα) is a type I interferon, an essential cytokine employed by the immune system to fight viruses. Although a number of the structures of type I interferons have been reported, most of the known structures of IFNα are in complex with its receptors. There are only two examples of structures of free IFNα: one is a dimeric X-ray structure without side-chain information; and another is an NMR structure of human IFNα. Although we have shown that Sortilin is involved in the secretion of IFNα, the details of the molecular interaction and the secretion mechanism remain unclear. Recently, we solved the X-ray structure of mouse Sortilin, but the structure of mouse IFNα remained unknown. In this study, we determined the crystal structure of mouse IFNα2 at 2.1 Å resolution and investigated its interaction with Sortilin. Docking simulations suggested that Arg22 of mouse IFNα2 is important for the interaction with mouse Sortilin. Mutation of Arg22 to alanine facilitated IFNα2 secretion, as determined by flow cytometry, highlighting the contribution of this residue to the interaction with Sortilin. These results suggest an important role for Arg22 in mouse IFNα for Sortilin-mediated IFNα trafficking.

Sortilin-derived peptides promote pancreatic beta-cell survival through CREB signaling pathway.

Deterioration of insulin secretion and pancreatic beta-cell mass by inflammatory attacks is one of the main pathophysiological features of type 2 diabetes (T2D). Therefore, preserving beta-cell mass and stimulating insulin secretion only in response to glucose for avoiding the hypoglycemia risks, are the most state-of-the-art option for the treatment of T2D. In this study we tested two correlated hypothesis that 1/ the endogenous peptide released from sortilin, known as PE, that stimulates insulin secretion only in response to glucose, protects beta-cells against death induced by cytokines, and 2/ Spadin and Mini-Spadin, two synthetic peptides derived from PE, that mimic the effects of PE in insulin secretion, also provide beneficial effect on beta-cells survival. We show that PE and its derivatives by inducing a rise of intracellular calcium concentration by depolarizing the membrane protect beta-cells against death induced by Interleukin-1ß. Using biochemical, confocal imaging and cell biology techniques, we reveal that the protective effects of PE and its derivatives rely on the activation of the CaM-Kinase pathway, and on the phosphorylation and activation of the transcription factor CREB. In addition, Mini-Spadin promotes beta-cell proliferation, suggesting its possible regenerative effect. This study highlights new possible roles of PE in pancreatic beta-cell survival and its derivatives as pharmacological tools against diabetes.

The multifaceted role of sortilin/neurotensin receptor 3 in human cancer development.

Sortilin (also known as neurotensin receptor 3) is a multitasking protein implicated in numerous pathophysiological processes, including cancer development, cardiovascular impairment, Alzheimer-type dementia, and depression. Although the definitive role of sortilin in human solid and hematological malignancies has been evidenced, few articles reviewed the task. The aim of the current review is to unravel the mechanisms by which sortilin controls oncogenicity and cancer progression; and also to summarize and discuss the original data obtained from international research laboratories on this topic. Questions on how sortilin is involving in the impairment of cell junctions, in exosomes composition and release, as well as in the regulation of epidermal growth factor receptor trafficking are also responded. In addition, we provide a special focus on the regulatory role of sortilin in signal transduction by either neurotrophins or neurotensin in normal and malignant cells. The relevance of sortilin with normal and cancer stem cells is also discussed. The last section provides a general overview of sortilin applications as a diagnostic and prognostic biomarker in the context of cancer detection. Finally, we comment on the future research aspects in which the field of cancer diagnosis, prognosis, and therapy might be developed.

Type II Binders Targeting the "GLR-Out" Conformation of the Pseudokinase STRADα.

Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.

Membrane packing defects in synaptic vesicles recruit complexin and synuclein.

Complexin-1 (Cpx) and α-synuclein (α-Syn) are involved in neurotransmitter release through an interaction with synaptic vesicles (SVs). Recent studies demonstrated that Cpx and α-Syn preferentially associate with highly curved membranes, like SVs, to correctly position them for fusion. Here, based on recent experimental results, to further propose a possible explanation for this mechanism, we performed in silico simulations probing interactions between Cpx or α-Syn and membranes of varying curvature. We found that the preferential association is attributed to smaller, curved membranes containing more packing defects that expose hydrophobic acyl tails, which may favorably interact with hydrophobic residues of Cpx and α-Syn. The number of membrane defects is proportional to the curvature and the size can be regulated by cholesterol.

Identification of Sortilin Alternatively Spliced Variants in Mouse 3T3L1 Adipocytes.

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein-protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort17b. Further, results demonstrate that over-expression of Sort17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.