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Multidrug-resistant Klebsiella pneumoniae (MDR-KP) infections have become a major global issue in the healthcare sector. Alternative viable tactics for combating bacterial infections, such as the use of bacteriophages, can be considered. One of the major challenges in phage therapy is the emergence of phage-resistant bacteria. This study isolated bacteriophages from water and soil samples against MDR-KP isolates. Susceptible bacterial hosts were exposed to phages at different concentrations and prolonged durations of time to obtain phage-resistant survivors. Phenotypic changes such as changes in growth rates, biofilm formation ability, antibiotic sensitivity patterns, and outer membrane proteins (OMPs) profiling of the survivors were studied. Our findings indicate that the phage ØKp11 and ØKp26 survivors had reduced growth rates and biofilm formation ability, altered antibiotic sensitivity patterns, and reduced OMPs expression compared with the parent MDR-KP002 isolate. These results suggest that the alternations in the bacterial envelope result in phenotypic phage resistance among MDR bacterial isolates. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01217-6.

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Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

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Human epidermal growth factor receptors (HER)-also known as EGFR or ErbB receptors-are a subfamily of receptor tyrosine kinases (RTKs) that play crucial roles in cell growth, division, and differentiation. HER4 (ErbB4) is the least studied member of this family, partly because its expression is lower in later stages of development. Recent work has suggested that HER4 can play a role in metastasis by regulating cell migration and invasiveness; however, unlike EGFR and HER2, the precise role that HER4 plays in tumorigenesis is still unresolved. Early work on HER family proteins suggested that there are direct interactions between the four members, but to date, there has been no single study of all four receptors in the same cell line with the same biophysical method. Here, we quantitatively measure the degree of association between HER4 and the other HER family proteins in live cells with a time-resolved fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is sensitive to the oligomerization state of membrane proteins in live cells, while simultaneously measuring single-cell protein expression levels and diffusion coefficients. Our PIE-FCCS results demonstrate that HER4 interacts directly with all HER family members in the cell plasma membrane. The interaction between HER4 and other HER family members intensified in the presence of a HER4-specific ligand. Our work suggests that HER4 is a preferred dimerization partner for all HER family proteins, even in the absence of ligands.

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Membrane protein-mediated resistance is a multidisciplinary challenge that spans fields such as medicine, agriculture, and environmental science. Understanding its complexity and devising innovative strategies are crucial for treating diseases like cancer and managing resistant pests in agriculture. This paper explores the dual nature of resistance mechanisms across different organisms: On one hand, animals, bacteria, fungi, plants, and insects exhibit convergent evolution, leading to the development of similar resistance mechanisms. On the other hand, influenced by diverse environmental pressures and structural differences among organisms, they also demonstrate divergent resistance characteristics. Membrane protein-mediated resistance mechanisms are prevalent across animals, bacteria, fungi, plants, and insects, reflecting their shared survival strategies evolved through convergent evolution to address similar survival challenges. However, variations in ecological environments and biological characteristics result in differing responses to resistance. Therefore, examining these differences not only enhances our understanding of adaptive resistance mechanisms but also provides crucial theoretical support and insights for addressing drug resistance and advancing pharmaceutical development.

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Peripheral membrane proteins (PMPs) are a subgroup of membrane-associated proteins that are water-soluble and bind to membranes, often reversibly, to perform their function. These proteins have been extensively studied in the aqueous state, but there is often a lack of high-resolution structural and functional studies of these proteins in the membrane-bound state. Currently, nuclear magnetic resonance (NMR) is among the best-equipped methods to study these relatively small proteins and domains, but current models have some disadvantages that prevent a full understanding of PMP interactions with membranes and lipids. Micelles, bicelles, and nanodiscs are all available for NMR observation but are based on synthetic lipids that may destabilize proteins or are too large to accommodate straightforward structural analysis. This protocol introduces a method for forming reverse micelles using lipids from natural sources, here called native reverse micelles. This technique allows the PMPs to embed within a shell of naturally derived lipids surrounding a small water core solubilized in an alkane solvent. PMP embedment in the lipid shell mimics binding to a cellular membrane. Here, naturally derived lipids from soy, bovine heart, and porcine brain are used in conjunction with n-dodecylphosphocholine (DPC) to encapsulate a PMP from either concentrated or dried protein, resulting in reverse micelles that may be confirmed via dynamic light scattering and NMR. This protocol allows for high-quality NMR data of PMPs interacting with membrane lipids within a biologically accurate environment. Key features • This protocol describes using natural lipids to construct reverse micelles for high-resolution NMR studies of proteins. • Initial optimization of encapsulation conditions proceeds through visual assessment, with dynamic light scattering (DLS) to measure size distribution, and NMR to observe protein behavior. • Membrane-interacting proteins are encapsulated in their membrane-bound state. Proteins that do not interact with membranes are housed in their water-solubilized state. • Structural, functional, and inhibitory studies may be performed on native reverse micelle-encapsulated proteins.

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Milk fat globule membrane (MFGM) is a three-layer membrane-like structure encasing natural milk fat globules (MFGs). MFGM holds promise as a nutritional supplement because of the numerous physiological functions of its constituent protein. This review summarizes and compares the differences in MFGM protein composition across various species, including bovines, goats, camels, mares, and donkeys, and different lactation periods, such as colostrum and mature milk, as assessed by techniques such as proteomics and mass spectrometry. We also discuss the health benefits of MFGM proteins throughout life. MFGM proteins promote intestinal development, neurodevelopment, and glucose and lipid metabolism by upregulating tight junction protein expression, brain function-related genes, and glucose and fatty acid biosynthesis processes. We focus on the mechanisms underlying these beneficial effects of MFGM proteins. MFGM proteins activate key substances in in signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, and myosin light chain kinase signaling pathways. Overall, the consumption of MFGM proteins plays an essential role in conferring health benefits, some of which are important throughout the mammalian life cycle.

Types and amounts of MFGM proteins in mammals, as assessed by proteomic and mass spectrometry analysis, are summarized.Colostrum MFGM contains more acute phase proteins, whereas mature milk has higher levels of mucins (1 and 15), ADPH, XDH, and FABP.Health benefits of MFGM proteins, including intestinal development, neurodevelopment, and immune activity enhancement, are summarized.MFGM proteins have been shown to significantly activate the PI3K/Akt/mTOR signaling pathway, promoting cell proliferation and glycolipid metabolism.

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Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the ERAD system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly-conserved ERAD system.

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Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

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X-ray diffraction crystallography has been most widely used for protein three-dimensional (3D) structure determination for which whether proteins are crystallizable is a central prerequisite. Yet, there are a number of procedures during protein crystallization, including protein material production, purification, and crystal production, which take turns affecting the crystallization outcome. Due to the expensive and laborious nature of this multi-stage process, various computational tools have been developed to predict protein crystallization propensity, which is then used to guide the experimental determination. In this study, we presented a novel deep learning framework, PLMC, to improve multi-stage protein crystallization propensity prediction by leveraging a pre-trained protein language model. To effectively train PLMC, two groups of features of each protein were integrated into a more comprehensive representation, including protein language embeddings from the large-scale protein sequence database and a handcrafted feature set consisting of physicochemical, sequence-based and disordered-related information. These features were further separately embedded for refinement, and then concatenated for the final prediction. Notably, our extensive benchmarking tests demonstrate that PLMC greatly outperforms other state-of-the-art methods by achieving AUC scores of 0.773, 0.893, and 0.913, respectively, at the aforementioned individual stages, and 0.982 at the final crystallization stage. Furthermore, PLMC is shown to be superior for predicting the crystallization of both globular and membrane proteins, as demonstrated by an AUC score of 0.991 for the latter. These results suggest the significant potential of PLMC in assisting researchers with the experimental design of crystallizable protein variants.

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Exosomes play a crucial role in various biological processes, such as human development, immune responses, and disease occurrence. The membrane proteins on exosomes are pivotal factors for their biological functionality. Currently, numerous membrane proteins have been identified on exosome membranes, participating in intercellular communication, mediating target cell recognition, and regulating immune processes. Furthermore, membrane proteins from exosomes derived from cancer cells can serve as relevant biomarkers for early cancer diagnosis. This article provides a comprehensive review of the composition of exosome membrane proteins and their diverse functions in the organism's biological processes. Through in-depth exploration of exosome membrane proteins, it is expected to offer essential foundations for the future development of novel biomedical diagnostics and therapies.

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Static electric fields play a considerable role in a variety of molecular nanosystems as diverse as single-molecule junctions, molecules supporting electrostatic catalysis, and biological cell membranes incorporating proteins. External electric fields can be applied to nanoscale samples with a conductive atomic force microscopy (AFM) probe in contact mode, but typically, no structural information is retrieved. Here we combine photothermal expansion infrared (IR) nanospectroscopy with electrostatic AFM probes to measure nanometric volumes where the IR field enhancement and the static electric field overlap spatially. We leverage the vibrational Stark effect in the polymer poly(methyl methacrylate) for calibrating the local electric field strength. In the relevant case of membrane protein bacteriorhodopsin, we observe electric-field-induced changes of the protein backbone conformation and residue protonation state. The proposed technique also has the potential to measure DC currents and IR spectra simultaneously, insofar enabling the monitoring of the possible interplay between charge transport and other effects.

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Prostate cancer (PCa) is one of the leading cancers in men and the lack of suitable biomarkers or their modulators results in poor prognosis. Membrane proteins (MPs) have a crucial role in the development and progression of PCa and can be attractive therapeutic targets. However, experimental limitations in targeting MPs hinder effective biomarker and inhibitor discovery. To overcome this barrier, computational methods can yield structural insights and screen large libraries of compounds, accelerating lead identification and optimization. In this review, we examine current breakthroughs in computer-aided drug design (CADD), with emphasis on structure-based approaches targeting the most relevant membrane-bound PCa biomarkers.

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Actin in neuronal processes is both stable and dynamic. The origin & functional roles of the different pools of actin is not well understood. We find that mutants that lack mitochondria, ric-7 and mtx-2; miro-1, in neuronal processes also lack dynamic actin. Mitochondria can regulate actin dynamics upto a distance ~80 µm along the neuronal process. Absence of axonal mitochondria and dynamic actin does not markedly alter the Spectrin Membrane Periodic Skeleton (MPS) in touch receptor neurons (TRNs). Restoring mitochondria inTRNs cell autonomously restores dynamic actin in a sod-2 dependent manner. We find that dynamic actin is necessary and sufficient for the localization of gap junction proteins in the TRNs and for the C. elegans gentle touch response. We identify an in vivo mechanism by which axonal mitochondria locally facilitate actin dynamics through reactive oxygen species that we show is necessary for electrical synapses & behaviour.

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Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.

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Cholesterol is a major component of plasma membranes and plays a significant role in actively regulating the functioning of several membrane proteins in humans. In this study, we focus on the role of cholesterol depletion on the voltage-gated sodium channel Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations revealed key dynamic changes of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity in the structural motifs linked to activation and fast-inactivation is observed, suggesting an easier transition of the channel between different gating states. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions at the functional level: Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results also point to cholesterol-driven changes of the geometry of drug-binding regions, hinting to a key role of the membrane environment in the regulation of drug effects.

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Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a ß-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the ß1- and the ß12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the ß1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the ß1-strand with the ß3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the ß12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the ß-signals of classical autotransporter proteins and outer membrane ß-barrel proteins in general. We therefore propose that the ß12-strand of YeeJ acts as its ß-signal and that the assembly of the YeeJ ß-barrel is driven by its C-terminal ß-strand, like in most other autotransporter proteins, despite its inverted domain organization.

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P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.

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Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein's native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelBSt), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis.

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The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three 'noNterm' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

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Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs.

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