Transmembrane Domain Of Protein Research Articles

Carbocation Footprinting of Soluble and Transmembrane Proteins

Here, we introduce carbocations (R3C+) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB+) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB+ increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo- and holo-myoglobin footprints shows that the TFB+ footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB+ is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB+ with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra- and trans-membrane domains of membrane proteins.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Leucine-rich repeats and immunoglobulin-like domains 1 deficiency affects hippocampal dendrite complexity and impairs cognitive function.

Leucine-rich repeat (LRR) transmembrane proteins have been directly linked to neurodevelopmental and cognitive disorders. We have previously shown that the LRR transmembrane protein, leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), is a physiological regulator of dendrite complexity of hippocampal pyramidal neurons and social behavior. In this study, we performed a battery of behavioral tests to evaluate spatial memory and cognitive capabilities in Lrig1 mutant mice. The cognitive assessment demonstrated deficits in recognition and spatial memory, evaluated by novel object recognition and object location tests. Moreover, we found that Lrig1-deficient mice present specific impairments in the processing of similar but not dissimilar locations in a spatial pattern separation task, which was correlated with an enhanced dendritic growth and branching of Doublecortin-positive immature granule cells of the dentate gyrus. Altogether, these findings indicate that Lrig1 plays an essential role in controlling morphological and functional plasticity in the hippocampus.

Abstract 1811: A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies

Abstract Immunotherapies through targeting of activating and inhibitory immune checkpoints (ICP) has revolutionized the treatment of cancers. To facilitate the translation of basic research into clinical application of these immunotherapies, we developed a pipeline of immunocompetent mouse models expressing humanized ICP instead of their mouse counterparts, thus compounds can be tested in the absence of endogenous cross-reacting mouse targets. The humanization strategy is target-dependent but ensures that the biology of the target, its physiological regulation and interacting partners are preserved. Herein, we describe a novel OX40 humanized mouse model (hOX40 model), designed to assess the efficacy and mechanism of action of compounds targeting human OX40 (hOX40). The expression of the fully human OX40 protein (human extracellular, transmembrane and intracellular domains) did not alter the distribution of immune cells in spleen, blood and bone marrow. hOX40 was expressed on CD4 and CD8 T cells upon activation and the kinetic of expression mirrored the expression of mouse OX40 in WT cells activated under the same conditions. Treg cells isolated from hOX40 mice expressed hOX40 and this expression was upregulated upon activation, also recapitulating the expression of mouse OX40 in WT cells, and suggesting that the regulation of OX40 expression was kept in the hOX40 mice. hOX40 was also found to be functional on CD4 T cells, as evidenced by the increased proliferation and cytokine production upon stimulation with human OX40 ligand. Importantly, hOX40 mice bearing MC38 tumors showed a tumor growth inhibition upon treatment with an anti-human OX40 therapy with agonist activity. This model should also enable the investigation of mechanism of action as well as tumor immunological memory, as a similar design of the hOX40 model was used for the assessment of a bispecific therapy targeting OX40 and CTLA-4 (Kvarnhammar et.al, 2019). The hOX40 mouse model is currently being crossed with other models expressing humanized immune checkpoint to enable the assessment of bispecifics and combo therapies. Citation Format: Fabiane Sônego, Gaëlle Martin, Audrey Beringer, Chloé Beuraud, Yacine Cherifi, Alexandre Fraichard, Patricia Isnard Petit, Kader Thiam. A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1811.

Looking for a safe haven: tail-anchored proteins and their membrane insertion pathways.

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.

Membrane protein biogenesis at the ER: the highways and byways.

The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.

Discovery and Characterization of a Novel Tomato mlo Mutant from an EMS Mutagenized Micro-Tom Population.

In tomato (Solanum lycopersicum), there are at least three SlMLO (Mildew resistance Locus O) genes acting as susceptibility genes for the powdery mildew disease caused by Oidium neolycopersici, namely SlMLO1, SlMLO5 and SlMLO8. Of the three homologs, the SlMLO1 gene plays a major role since a natural mutant allele called ol-2 can almost completely prevent fungal penetration by formation of papillae. The ol-2 allele contains a 19-bp deletion in the coding sequence of the SlMLO1 gene, resulting in a premature stop codon within the second cytoplasmic loop of the predicted protein. In this study, we have developed a new genetic resource (M200) in the tomato cv. Micro-Tom genetic background by means of ethyl methane sulfonate (EMS) mutagenesis. The mutant M200 containing a novel allele (the m200 allele) of the tomato SlMLO1 gene showed profound resistance against powdery mildew with no fungal sporulation. Compared to the coding sequence of the SlMLO1 gene, the m200 allele carries a point mutation at T65A. The SNP results in a premature stop codon L22* located in the first transmembrane domain of the complete SlMLO1 protein. The length of the predicted protein is 21 amino acids, while the SlMLO1 full-length protein is 513 amino acids. A high-resolution melting (HRM) marker was developed to distinguish the mutated m200 allele from the SlMLO1 allele in backcross populations. The mutant allele conferred recessive resistance that was associated with papillae formation at fungal penetration sites of plant epidermal cells. A comprehensive list of known mlo mutations found in natural and artificial mutants is presented, which serves as a particularly valuable resource for powdery mildew resistance breeding.

Synthesis of SARS‐CoV‐2 Transmembrane Domains Using Solid Phase Peptide Chemistry and Analysis of Their Oligomerization for Novel Drug Design

The SARS‐CoV‐2 spike protein transmembrane (TM) domain is an important component of the protein that contributes to its oligomerization for its active configuration. An understanding of this function can yield valuable data to future research on how to inhibit the virus’ infection of a host. The TM region of the protein is not as well studied due to the complication that nonpolar peptides can have in large groups; therefore, an understanding of this region could lead to novel discoveries. A peptide of the TM domain must be synthesized by using Solid Phase Peptide Synthesis, in which a resin is utilized to build a peptide off the c‐terminus using Fmoc‐protected amino acids and a strong base. A fluorescence assay can be performed to determine its oligomerization state by looking at the change of fluorescence that occurs when a quencher is added to the solution. Once the oligomerization state of the spike proteins TM domain is determined, this information will be beneficial the development of drugs against the virus. The set up to run this synthesis was developed in the lab and is causing premature termination of the amino acid chain. Current work involves addressing a potential issue of hydrophobicity due to the volume non‐polar residues, which is currently being address by trying a TGR resin that has a polystyrene core which is suited for long peptides. It is anticipated that addressing this issue will lead to a peptide that can be purified. The future implications of this research is that it has the potential of being useful for drugs that would inhibit the active configuration of SARS‐CoV‐2.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Transmembrane domains of type III-secreted proteins affect bacterial-host interactions in enteropathogenic E. coli

ABSTRACT Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.

Dissecting Role of Charged Residue from Transmembrane Domain 5 of Latent Membrane Protein 1 via In Silico Simulations and Wet-Lab Experiments.

Charged residues are frequently found in the transmembrane segments of membrane proteins, which reside in the hydrophobic bilayer environment. Charged residues are critical for the function of membrane protein. However, studies of their role in protein oligomerization are limited. By taking the fifth transmembrane domain (TMD5) of latent membrane protein 1 from the Epstein-Barr virus as a prototype model, in silico simulations and wet-lab experiments were performed to investigate how the charged states affect transmembrane domain oligomerization. Molecular dynamics (MD) simulations showed that the D150-protonated TMD5 trimer was stable, whereas unprotonated D150 created bends in the helices which distort the trimeric structure. D150 was mutated to asparagine to mimic the protonated D150 in TMD5, and the MD simulations of different D150N TMD5 trimers supported that the protonation state of D150 was critical for the trimerization of TMD5. In silico mutations found that D150N TMD5 preferred to interact with TMD5 to form the heterotrimer (1 D150N TMD5:2 protonated TMD5s) rather than the heterotrimer (2 D150N TMD5s:1 protonated TMD5). D150R TMD5 interacted with TMD5 to form the heterotrimer (1 D150R TMD5:2 protonated TMD5). These in silico results imply that D150N TMD5 and D150R TMD5 peptides may be probes for disrupting TMD5 trimerization, which was supported by the dominant-negative ToxR assay in bacterial membranes. In all, this study elucidates the role of charged residues at the membrane milieu in membrane protein oligomerization and provides insight into the development of oligomerization-regulating peptides for modulating transmembrane domain lateral interactions.

Loss of the vaccinia virus 35-amino acid hydrophobic O3 protein is partially compensated by mutations in the transmembrane domains of other entry proteins.

Eleven highly conserved proteins comprise the poxvirus entry-fusion complex (EFC). We focused on vaccinia virus (VACV) O3, a 35-amino acid, largely hydrophobic component of unknown specific function. Experimental evolution was carried out by blindly passaging a virus that was severely impaired in entry due to deletion of the gene encoding O3. Large plaque variants that arose spontaneously were discerned by round four and their numbers increased thereafter. Genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. There were frame-shift mutations in consecutive Ts in open reading frames F9L and D8L and a nonsynonymous base substitution in L5R. F9 and L5 are EFC proteins and D8 is involved in VACV cell attachment. The F9L mutation occurred by round four in each of three independant passages, whereas the L5R and D8L mutations were detected only after nearly all of the genomes already had the F9L mutation. Viruses with deletions of O3L and single or double F9L, L5R and D8L mutations were constructed by homologous recombination. In a single round of infection, viruses with adaptive mutations including F9L alone or in combination exhibited statistically significant higher virus titers than the parental O3L deletion mutant or the L5R or D8L mutants, consistent with the order of selection during the passages. Further analyses indicated that the adaptive F9L mutants also had higher infectivities, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3.IMPORTANCE Entry into cells is an essential first step in virus replication and an important target of vaccine- elicited immunity. For enveloped viruses, this step involves the fusion of viral and host membranes to form a pore allowing entry of the genome and associated proteins. Poxviruses are unique in that this function is mediated by an entry-fusion complex (EFC) of eleven transmembrane proteins rather than by one or a few. The large number of proteins has hindered investigation of their individual roles. We focused on O3, a predominantly hydrophobic 35 amino acid component of the vaccinia virus EFC, and found that spontaneous mutations in the transmembrane domains of certain other entry proteins can partially compensate for the absence of O3. The mutants exhibited increased infectivity, entry and assembly or stability of the EFC.

Role of key amino acids in the transmembrane domain of the Newcastle disease virus fusion protein.

Newcastle disease (ND), caused by the Newcastle disease virus (NDV), is transmitted by poultry with severe infectivity and a high fatality rate. The fusion (F) protein on the NDV envelope facilitates the merger of the viral and host cell membranes with the help of the homologous hemagglutinin-neuraminidase protein (HN). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion, but the key amino acids in NDV F TM domains have not been identified. Site-directed mutagenesis was utilized to change the conserved amino acids at 500, 501, 502, 505, 510, 513, 516, 519, and 520 to alanine. It was found that mutants L519 and V520 had an interrupted protein expression, decreased to below 10%, and mutants A500, I505, V513, and V516 had a hypoactive impact on fusion activity, decreased to 85.38%, 67.05%, 55.38% and 51.13% of wt F, respectively. The results indicated that the TM domain plays a vital part in the fusion activity of the NDV F protein.

Cloning, Spatiotemporal Expression and Prokaryotic Expression of ACC Oxidase Gene (<I>BnACO2</i>) in Ramie

ACC oxidase (ACO) is a key enzyme in ethylene synthesis pathway. This study by rt-pcr with RACE technology get a ACO gene cloning, named  BnACO2 , total length of the gene cDNA sequence is 1 377 bp, the open reading frame of 957 bp, coded 318 amino acid polypeptide, predict its molecular weight and isoelectric point (pl) 36.145 kD and 5.60, respectively, bioinformatics analysis showed that  BnACO2  no encoding protein signal peptide and transmembrane domain structure, subcellular localization in the cytoplasm. The similarity of ACO gene nucleotide sequence and amino acid sequence was more than 82% and 84% respectively. Phylogenetic analysis showed that the ACO gene was closely related to jute and hemp. Real-time fluorescence quantitative PCR analysis showed that  BnACO2  gene was expressed in all parts of ramie, especially in female flower buds. The prokaryotic expression vector PQe- BnACO2  was successfully constructed by double enzyme digestion. At 37°C, 1.0mmol/L IPTG induced PQe- BnACO2  to express the target protein, and the molecular weight of the induced protein was about 36.15kda, which was consistent with the predicted protein size. This lays the foundation for further study on the function of  BnACO2  gene.

An in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.

Altered Hinge Conformations in APP Transmembrane Helix Mutants May Affect Enzyme-Substrate Interactions of γ-Secretase.

Cleavage of substrates by γ-secretase is an inherently slow process where substrate-enzyme affinities cannot be broken down into specific sequence requirements in contrast to soluble proteases. Nevertheless, despite its apparent sequence tolerance single point mutations in amyloid precursor protein can severely affect cleavage efficiencies and change product line preferences. We have determined by NMR spectroscopy the structures of the transmembrane domain of amyloid precursor protein in TFE/water and compared it to that of four mutants: two FAD mutants, V44M and I45T, and the two diglycine hinge mutants, G38L and G38P. In accordance with previous publications, the transmembrane domain is composed of two helical segments connected by the diglycine hinge. Mutations alter kink angles and structural flexibility. Furthermore, to our surprise, we observe different, but specific mutual orientations of N- and C-terminal helical segments in the four mutants compared to the wildtype. We speculate that the observed orientations for G38L, G38P, V44M, and I45T lead to unfavorable interactions with γ-secretase exosites during substrate movement to the enzyme's active site in presenilin and/or for the accommodation into the substrate-binding cavity of presenilin.

Cell scientist to watch – Hayley Sharpe

ABSTRACT Hayley Sharpe graduated from the University of Bath, UK, with a degree in biochemistry. She then joined the laboratory of Sean Munro for her PhD in cell and molecular biology at the Medical Research Council Laboratory of Molecular Biology (MRC-LMB) in Cambridge, UK. There, she investigated the properties of protein transmembrane domains embedded within the organelle membranes of the secretory pathway. She next moved to San Francisco, USA, for postdoctoral work at Genentech, where she studied clinical resistance mechanisms to a Hedgehog pathway inhibitor in skin cancer. In 2016, Hayley returned to the UK to establish her own research group at the Cambridge Institute for Medical Research, and in 2019, she moved to the Babraham Institute, also in Cambridge. Her laboratory works on understanding how protein tyrosine phosphatases function in signalling and cell–cell communication, with a particular focus on a family of cell surface receptors. Hayley became part of the EMBO Young Investigator Programme in 2019 and was awarded the Lister Institute Research Prize in 2020.

Mapping bilayer thickness in the ER membrane.

In the plasma membrane and in synthetic membranes, resident lipids may laterally unmix to form domains of distinct biophysical properties. Whether lipids also drive the lateral organization of intracellular membranes is largely unknown. Here, we describe genetically encoded fluorescent reporters visualizing local variations in bilayer thickness. Using them, we demonstrate that long-chained ceramides promote the formation of discrete domains of increased bilayer thickness in the yeast ER, particularly in the future plane of cleavage and at ER-trans-Golgi contact sites. Thickening of the ER membrane in the cleavage plane contributed to the formation of lateral diffusion barriers, which restricted the passage of short, but not long, protein transmembrane domains between the mother and bud ER compartments. Together, our data establish that the ER membrane is laterally organized and that ceramides drive this process, and provide insights into the physical nature and biophysical mechanisms of the lateral diffusion barriers that compartmentalize the ER.