Membrane Proteins Research Articles

TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Structure and Substrate Recognition of the Escherichia coli Transport Protein NupG from the Nucleoside: H+ Symporter (NHS) Family

The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N- and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).

SMIM1 absence is associated with reduced energy expenditure and excess weight.

Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Anesthetic Mechanisms: Synergistic Interactions With Lipid Rafts and Voltage-Gated Sodium Channels.

Despite successfully utilizing anesthetics for over 150 years, the mechanism of action remains relatively unknown. Recent studies have shown promising results, but due to the complex interactions between anesthetics and their targets, there remains a clear need for further mechanistic research. We know that lipophilicity is directly connected to anesthetic potency since lipid solubility relates to anesthetic partition into the membrane. However, clinically relevant concentrations of anesthetics do not significantly affect lipid bilayers but continue to influence various molecular targets. Lipid rafts are derived from liquid-ordered phases of the plasma membrane that contain increased concentrations of cholesterol and sphingomyelin and act as staging platforms for membrane proteins, including ion channels. Although anesthetics do not perturb membranes at clinically relevant concentrations, they have recently been shown to target lipid rafts. In this review, we summarize current research on how different types of anesthetics-local, inhalational, and intravenous-bind and affect both lipid rafts and voltage-gated sodium channels, one of their major targets, and how those effects synergize to cause anesthesia and analgesia. Local anesthetics block voltage-gated sodium channel pores while also disrupting lipid packing in ordered membranes. Inhalational anesthetics bind to the channel pore and the voltage-sensing domain while causing an increase in the number, size, and diameter of lipid rafts. Intravenous anesthetics bind to the channel primarily at the voltage-sensing domain and the selectivity filter, while causing lipid raft perturbation. These changes in lipid nanodomain structure possibly give proteins access to substrates that have translocated as a result of these structural alterations, resulting in lipid-driven anesthesia. Overall, anesthetics can impact channel activity either through direct interaction with the channel, indirectly through the lipid raft, or both. Together, these result in decreased sodium ion flux into the cell, disrupting action potentials and producing anesthetic effects. However, more research is needed to elucidate the indirect mechanisms associated with channel disruption through the lipid raft, as not much is known about anionic lipid products and their influence over voltage-gated sodium channels. Anesthetics' effect on S-palmitoylation, a promising mechanism for direct and indirect influence over voltage-gated sodium channels, is another auspicious avenue of research. Understanding the mechanisms of different types of anesthetics will allow anesthesiologists greater flexibility and more specificity when treating patients.

Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.

Isaridin E Protects against Sepsis by Inhibiting Von Willebrand Factor-Induced Endothelial Hyperpermeability and Platelet-Endothelium Interaction.

Endothelial hyperpermeability is pivotal in sepsis-associated multi-organ dysfunction. Increased von Willebrand factor (vWF) plasma levels, stemming from activated platelets and endothelium injury during sepsis, can bind to integrin αvβ3, exacerbating endothelial permeability. Hence, targeting this pathway presents a potential therapeutic avenue for sepsis. Recently, we identified isaridin E (ISE), a marine-derived fungal cyclohexadepsipeptide, as a promising antiplatelet and antithrombotic agent with a low bleeding risk. ISE's influence on septic mortality and sepsis-induced lung injury in a mouse model of sepsis, induced by caecal ligation and puncture, is investigated in this study. ISE dose-dependently improved survival rates, mitigating lung injury, thrombocytopenia, pulmonary endothelial permeability, and vascular inflammation in the mouse model. ISE markedly curtailed vWF release from activated platelets in septic mice by suppressing vesicle-associated membrane protein 8 and soluble N-ethylmaleide-sensitive factor attachment protein 23 overexpression. Moreover, ISE inhibited healthy human platelet adhesion to cultured lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), thereby significantly decreasing vWF secretion and endothelial hyperpermeability. Using cilengitide, a selective integrin αvβ3 inhibitor, it was found that ISE can improve endothelial hyperpermeability by inhibiting vWF binding to αvβ3. Activation of the integrin αvβ3-FAK/Src pathway likely underlies vWF-induced endothelial dysfunction in sepsis. In conclusion, ISE protects against sepsis by inhibiting endothelial hyperpermeability and platelet-endothelium interactions.

Acute myeloid leukemia stem cell signature gene EMP1 is not an eligible therapeutic target.

Relapse in pediatric acute myeloid leukemia (pedAML) patients is known to be associated with residual leukemic stem cells (LSC). We have previously shown that epithelial membrane protein 1 (EMP1) is significantly overexpressed in LSC compared to hematological stem cell fractions. EMP1 was also documented as part of the 17-gene stemness score and a 6-membrane protein gene score, both correlating high EMP1 expression with worse overall survival. However, its potential as a therapeutic target in pedAML is still unexplored. Association analyses of EMP1 expression with clinical and molecular AML characteristics were performed. Expression of EMP1 was evaluated in pedAML and cord blood samples. Expression in normal blood cells and tissues was evaluated by flow cytometry and immunohistochemistry, respectively. In silico analyses showed variable mRNA expression of EMP1 in multiple pedAML datasets, and a significant correlation between high EMP1 transcript levels and the presence of inv(16). Flow cytometry showed overexpression of EMP1 in pedAML samples, as well as expression in normal blood subsets. Importantly, immunohistochemistry revealed EMP1 expression in multiple normal tissues. Although EMP1 presents as an interesting membrane-associated target in pedAML, its abundant expression in normal blood cells and tissues will impede it from further exploration as a therapeutic target. EMP1 is highly expressed in multiple cancer types, but expression in acute myeloid leukemia (AML) and normal tissues is unexplored. As EMP1 is investigated in other cancer types, expression in normal tissues and blood cells is relevant in predicting the success of EMP1-targeted therapies. In this study, we showed expression of EMP1 in multiple tissues, predicting high on-target off-tumor toxicity, which will warn other researchers of possible toxicities when generating EMP1-targeted therapy. Finally, we showed that high EMP1 expression is associated with better overall survival of pediatric AML patients, reducing the need for EMP1-targeted therapy.

Three-Dimensional Interaction Homology: Deconstructing Residue-Residue and Residue-Lipid Interactions in Membrane Proteins.

A method is described to deconstruct the network of hydropathic interactions within and between a protein's sidechain and its environment into residue-based three-dimensional maps. These maps encode favorable and unfavorable hydrophobic and polar interactions, in terms of spatial positions for optimal interactions, relative interaction strength, as well as character. In addition, these maps are backbone angle-dependent. After map calculation and clustering, a finite number of unique residue sidechain interaction maps exist for each backbone conformation, with the number related to the residue's size and interaction complexity. Structures for soluble proteins (~749,000 residues) and membrane proteins (~387,000 residues) were analyzed, with the latter group being subdivided into three subsets related to the residue's position in the membrane protein: soluble domain, core-facing transmembrane domain, and lipid-facing transmembrane domain. This work suggests that maps representing residue types and their backbone conformation can be reassembled to optimize the medium-to-high resolution details of a protein structure. In particular, the information encoded in maps constructed from the lipid-facing transmembrane residues appears to paint a clear picture of the protein-lipid interactions that are difficult to obtain experimentally.

Diastereomers of Coibamide A Show Altered Sec61 Client Selectivity and Ligand-Dependent Activity against Patient-Derived Glioma Stem-like Cells.

Coibamide A (CbA) is a cyanobacterial lariat depsipeptide that selectively inhibits multiple secreted and integral membrane proteins from entering the endoplasmic reticulum secretory pathway through binding the alpha subunit of the Sec61 translocon. As a complex peptide-based macrocycle with 13 stereogenic centers, CbA is presumed to adopt a conformationally restricted orientation in the ligand-bound state, resulting in potent antitumor and antiangiogenic bioactivity. A stereochemical structure-activity relationship for CbA was previously defined based on cytotoxicity against established cancer cell lines. However, the ability of synthetic isomers to inhibit the biosynthesis of specific Sec61 substrates was unknown. Here, we report that two less toxic diastereomers of CbA, [L-Hiv2]-CbA and [L-Hiv2, L-MeAla11]-CbA, are pharmacologically active Sec61 inhibitors. Both compounds inhibited the expression of a secreted reporter (Gaussia luciferase), VEGF-A, and a Type 1 membrane protein (VCAM1), while [L-Hiv2]-CbA also decreased the expression of ICAM1 and BiP/GRP78. Analysis of 43 different chemokines in the secretome of SF-268 glioblastoma cells revealed different inhibitory profiles for the two diastereomers. When the cytotoxic potential of CbA compounds was compared against a panel of patient-derived glioblastoma stem-like cells (GSCs), Sec61 inhibitors were remarkably toxic to five of the six GSCs tested. Each ligand showed a distinct cytotoxic potency and selectivity pattern for CbA-sensitive GSCs, with IC50 values ranging from subnanomolar to low micromolar concentrations. Together, these findings highlight the extreme sensitivity of GSCs to Sec61 modulation and the importance of ligand stereochemistry in determining the spectrum of inhibited Sec61 client proteins.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatographyremains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G])did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

A larger TatBC complex associates with TatA clusters for transport of folded proteins across the bacterial cytoplasmic membrane

The twin-arginine translocation (Tat) system transports folded proteins across energized biological membranes in bacteria, plastids, and plant mitochondria. In Escherichia coli, the three membrane proteins TatA, TatB and TatC associate to enable Tat transport. While TatB and TatC together form complexes that bind Tat-dependently transported proteins, the TatA component is responsible for the permeabilization of the membrane during transport. With wild type Tat systems, the TatB- and TatC-containing Tat complexes TC1 and TC2 can be differentiated. Their TatA content has not been resolved, nor could they be assigned to any step of the translocation mechanism. It is therefore a key question of current Tat research to understand how TatA associates with Tat systems during transport. By analyzing affinity-purified Tat complexes with mutations in TatC that selectively enrich either TC1 or TC2, we now for the first time demonstrate that both Tat complexes associate with TatA, but the larger TC2 recruits significantly more TatA than the smaller TC1. Most TatA co-purified as multimeric clusters. Using site-specific photo cross-linking, we could detect TatA–TatC interactions only near TatC transmembrane helices 5 and 6. Substrate-binding did not change the interacting positions but affected the stability of the interaction, pointing to a substrate-induced conformational transition. Together, our findings indicate that TatA clusters associate with TatBC without being integrated into the complex by major rearrangements. The increased TatA affinity of the larger Tat complex TC2 suggests that functional assembly is advanced in this complex.

ATP-Binding Cassette Transporter of Clinical Significance: Sideroblastic Anemia.

The ATP-binding cassette (ABC) transporters are a vast group of 48 membrane proteins, some of which are of notable physiological and clinical importance. Some ABC transporters are involved in functions such as the transport of chloride ions, bilirubin, reproductive hormones, cholesterol, and iron. Consequently, genetic or physiological disruption in these functions is manifested in various disease processes like cystic fibrosis, Tangier disease, and sideroblastic anemia. Among other etiologies, primary sideroblastic anemia results from a genetic mutation in the ATP-binding cassette-7 (ABCB7), a member of the ABC transporter family. There are not many articles specifically tackling the disease processes caused by ABC transporters in detail. Some testing methodologies previously reported in the available literature for investigating sideroblastic anemia need updating. Here, we expound on the relevance of ABCB7 as a clinically important ABC transporter and a rare participant in the disease process of Sideroblastic anemia. The other genetic and secondary etiologies of sideroblastic anemia, which do not involve mutations in the ABCB7 protein, are also described. We review the pathophysiology, clinical course, symptoms, diagnosis, and treatment of sideroblastic anemia with a focus on modern technologies for laboratory testing.

Ankyrin-B is required for the establishment and maintenance of lens cytoarchitecture, mechanics, and clarity.

This study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

A distinct dimer configuration of a diatom Get3 forming a tetrameric complex with its tail-anchored membrane cargo

BackgroundMost tail-anchored (TA) membrane proteins are delivered to the endoplasmic reticulum through a conserved posttranslational pathway. Although core mechanisms underlying the targeting and insertion of TA proteins are well established in eukaryotes, their role in mediating TA protein biogenesis in plants remains unclear. We reported the crystal structures of algal arsenite transporter 1 (ArsA1), which possesses an approximately 80-kDa monomeric architecture and carries chloroplast-localized TA proteins. However, the mechanistic basis of ArsA2, a Get3 (guided entry of TA proteins 3) homolog in plants, for TA recognition remains unknown.ResultsHere, for the first time, we present the crystal structures of the diatom Pt-Get3a that forms a distinct ellipsoid-shaped tetramer in the open (nucleotide-bound) state through crystal packing. Pulldown assay results revealed that only tetrameric Pt-Get3a can bind to TA proteins. The lack of the conserved zinc-coordination CXXC motif in Pt-Get3a potentially leads to the spontaneous formation of a distinct parallelogram-shaped dimeric conformation in solution, suggesting a new dimer state for subsequent tetramerization upon TA targeting. Pt-Get3a nonspecifically binds to different subsets of TA substrates due to the lower hydrophobicity of its α-helical subdomain, which is implicated in TA recognition.ConclusionsOur study provides new insights into the mechanisms underlying TA protein shielding by tetrameric Get3 during targeting to the diatom’s cell membrane.

Protein Binder Toolbox for Studies of Solute Carrier Transporters

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Advances in Chlamydia trachomatis Vaccination: Unveiling the Potential of Major Outer Membrane Protein Derivative Constructs.

Chlamydia (C.) trachomatis, a leading cause of sexually transmitted infections (STIs) worldwide, continues to be a significant public health concern. The majority of infections are asymptomatic and, when left untreated, severe sequelae such as infertility and chronic pelvic pain can occur. Despite decades of research, an effective vaccine remains elusive. This review focuses on the potential of Major Outer Membrane Protein (MOMP)-derived constructs as promising candidates for C. trachomatis vaccination. MOMP, the most abundant protein in the outer membrane of C. trachomatis, has been a focal point of vaccine research over the years due to its antigenic properties. To overcome issues associated with the use of full MOMP as a vaccine antigen, derivative constructs have been studied. As these constructs are often not sufficiently immunogenic, antigen delivery systems or accompanying adjuvants are required. Additionally, several immunization routes have been explored with these MOMP-derived vaccine antigens, and determining the optimal route remains an ongoing area of research. Future directions and challenges in the field of C. trachomatis vaccination are discussed.

Determining the minimal amount of DMSO necessary to stabilize the Angiomotin lipid binding domain

Angiomotins (Amots) are a family of adaptor proteins with important roles in cell growth, migration, and proliferation. The Amot coiled-coil homology (ACCH) domain, has a high affinity for non-phosphorylated phosphatidylinositol and mono-phosphorylated phosphatidylinositol. This provides specificity in the membrane association. This membrane specificity is linked with the targeting and recycling of the membrane protein in order to maintain normal cell phenotypes and function. Therefore, we endeavored to understand the protein function by studying the atomic structure of the protein. Our laboratory looked to determine the structure using nuclear magnetic resonance (NMR), which requires higher protein concentrations than those possible in our current buffered solutions. Based on literature reported on other proteins, DMSO can be used as a stabilizing agent up to 33-70% DMSO. Higher concentrations have led DMSO to become a denaturing agent. Therefore, this work shows our preliminary findings for the minimal amount of dimethyl sulfoxide (DMSO) needed to stabilize the domain at higher concentrations without disrupting its native structure. To that end, we determined DMSO related changes in protein structure seen as shifts in the melting temperature that is determined by dynamic scanning fluorescence measurements. As a result, we found that the ACCH domain is denatured in solutions >10% DMSO.