Membrane Proteins Research Articles

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

Type I toxin-antitoxin systems in bacteria: from regulation to biological functions.

Toxin-antitoxin systems are ubiquitous in the prokaryotic world and widely distributed among chromosomes and mobile genetic elements. Several different toxin-antitoxin system types exist, but what they all have in common is that toxin activity is prevented by the cognate antitoxin. In type I toxin-antitoxin systems, toxin production is controlled by an RNA antitoxin and by structural features inherent to the toxin messenger RNA. Most type I toxins are small membrane proteins that display a variety of cellular effects. While originally discovered as modules that stabilize plasmids, chromosomal type I toxin-antitoxin systems may also stabilize prophages, or serve important functions upon certain stress conditions and contribute to population-wide survival strategies. Here, we will describe the intricate RNA-based regulation of type I toxin-antitoxin systems and discuss their potential biological functions.

The Role of Lipid Intrinsic Curvature in the Droplet Interface Bilayer.

Model bilayers are constructed from lipids having different intrinsic curvatures using the droplet interface bilayer (DIB) method, and their static physicochemical properties are determined. Geometrical and tensiometric measurements are used to derive the free energy of formation (ΔF) of a two-droplet DIB relative to a pair of isolated aqueous droplets, each decorated with a phospholipid monolayer. The lipid molecules employed have different headgroup sizes but identical hydrophobic tail structure, and each is characterized by an intrinsic curvature value (c0) that increases in absolute value with decreasing size of headgroup. Mixtures of lipids at different ratios were also investigated. The role of curvature stress on the values of ΔF of the respective lipid bilayers in these model membranes is discussed and is illuminated by the observation of a decrement in ΔF that scales as a near linear function of c02. Overall, the results reveal an association that should prove useful in studies of ion channels and other membrane proteins embedded in model droplet bilayer systems that will impact the understanding of protein function in cellular membranes composed of lipids of high and low curvature.

VEGF-R2/CAV-1 Interaction Induced by Resveratrol/Eicosapentaenoic Acid/Docosahexaenoic Acid-Enriched Formulation through Functional Detergent-Resistant Membranes Is Associated with Decreased VEGF-A Release in ARPE-19 Cells.

Omega-3 fatty acids (O3FAs) and resveratrol (RSV) are known to be beneficial for certain eye diseases, such as age-related macular degeneration (AMD). Neovascular AMD is characterized by abnormal blood vessel formation due to the excessive synthesis of vascular endothelial growth factor (VEGF) by retinal pigment epithelium (RPE) cells. The study investigates whether a formulation based on eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and RSV is capable of counteracting VEGF-A secretion, and elucidates the molecular mechanism. The study finds, using ELISA, that O3FAs/RSV reduces VEGF-A secretion in human RPE cells. This phenomenon is related to the increased interaction between VEGF-receptor 2 (VEGF-R2) and caveolin-1 (CAV-1), a protein of detergent-resistant membranes (DRMs), as determined by co-immunoprecipitation and proximity ligation assay. Using microscale thermophoresis, the study confirms that O3FAs/RSV causes a high-affinity interaction. Isolation and analysis of DRMs reveal that this interaction is concomitant with VEGF-R2 relocalization in DRMs. The depletion of DRMs by a cholesterol-chelating agent blocks the VEGF-R2/CAV-1 interaction and EPA/DHA/RSV-mediated impairment of VEGF production. This specific interaction can provide a new strategy for countering VEGF-A production in human RPE cells and, consequently, reducing neovascularization in AMD. Further preclinical studies involving O3FAs and polyphenols are warranted.

Erythrocyte membrane-camouflaged mesoporous silica composite nanoparticles for loading and controlled release of the hepatoprotective agent silibinin: A-synthesis and physicochemical characterization.

Milk thistle has long been used in the treatment of liver and biliary disorders. In the present study, to make a long-acting delivery system for silibinin (SBN, a major active constituent of milk thistle seeds with antioxidant and hepatoprotective function), mesoporous silica composite nanoparticles (NC) were synthesized and coated with RBC membrane. A modified Stöber method was used for NC synthesis, which was then characterized using FE-SEM, DLS, TEM, FTIR, and EDAX techniques. A suitable lysis buffer was used to prepare RBC-ghost, and sonication was used to coat SBN-loaded NC (SBN-NC). The RBC-ghost coated SBN-NC (SBN-NC-RBCG) was evaluated by SDS-PAGE, Bradford, TEM, EDAX, and DLS methods. SBN release was then compared for the SBN-NC and SBN-NC-RBCG samples. the RBC membrane proteins were recovered from the coating of SBN-NC-RBCG, and SBN release was sustained over 24h when compared with the SBN-NC. Overall, through prolonging circulation in the bloodstream and evading the immune system, the developed system can improve SBN bioavailability in liver inflammation and fibrosis conditions that need further research.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

NMDA receptor autoantibodies primarily impair the extrasynaptic compartment.

Autoantibodies directed against the N-methyl-D-aspartate receptor (NMDAR-Ab) are pathogenic immunoglobulins detected in patients suffering from NMDAR encephalitis. NMDAR-Ab alter the receptor membrane trafficking, synaptic transmission and neuronal network properties, leading to patients' neurological and psychiatric symptoms. Patients often have very little neuronal damage but rapid and massive (treatment-responsive) brain dysfunctions related to unknown early mechanism of NMDAR-Ab. Our understanding of this early molecular cascade remains surprisingly fragmented. Here, we used a combination of single molecule-based imaging of membrane proteins to unveil the spatio-temporal action of NMDAR-Ab onto live hippocampal neurons. We first demonstrate that different clones of NMDAR-Ab primarily affect extrasynaptic -and not synaptic- NMDAR. In the first minutes, NMDAR-Ab increase extrasynaptic NMDAR membrane dynamics, de-clustering its surface interactome. NMDAR-Ab also rapidly reshuffle all membrane proteins located at the extrasynaptic compartment. Consistent with this alteration of multiple proteins, NMDAR-Ab effects were not mediated through the sole interaction between NMDAR and EphB2 receptor. At the long-term, NMDAR-Ab reduce NMDAR synaptic pool by slowing down receptor membrane dynamics in a cross-linking independent manner. Remarkably, exposing only extrasynaptic NMDAR to NMDAR-Ab was sufficient to produce their full-blown effect on synaptic receptors. Collectively, we demonstrate that NMDAR-Ab first impair extrasynaptic proteins, and then the synaptic ones. These data shed thus new, and unsuspected, lights on the mode of action of NMDAR-Ab and likely to our understanding of (extra)synaptopathies.

Autophagy initiation triggers p150Glued-AP-2β interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus.

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.

Hydrophobic Mismatch in the Thylakoid Membrane Regulates Photosynthetic Light Harvesting.

The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.

Getting membrane proteins into shape

In this issue, Ji etal.1 show how a multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase.

Tissue clearing and three-dimensional imaging of intact tissues: a review on FACT protocol.

FACT is a developed technique for clearing tissues that does not use acrylamide. Since the removal of lipids is crucial for transparency and efficient antibody staining throughout the tissue, especially for microscopy and imaging, it is a harmful process that can cause the loss of important biological molecules such as proteins. The FACT technique overcomes this by chemically bonding the membrane and intracellular proteins with the extracellular matrix, creating a massive 3D hydrogel matrix and providing structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, the FACT technique requires less labor and harmful chemicals and is therefore considered a more suitable option. In this study, we describe the complete FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving structure and improving image quality. The protocol includes tissue perfusion, fixation, clearing, antibody staining, refractive index matching (RIM) (), microscopy, and imaging. The timing for each step varies depending on the size, weight, and type of tissue, as well as the type of immunostaining. We provide an example of the FACT protocol using mouse brain tissue, which demonstrates its suitability for molecular interrogation analysis of large tissues. The FACT technique has been successfully performed on different types of tissues, making it a favorable choice for a variety of applications.

Impaired SHH responsiveness of induced pluripotent stem cell-derived floor plate cells carrying the LRRK2 I1371V mutation contributes to the ontogenic origin of lower dopaminergic-neuron yield.

Lower population of dopaminergic (DA)-neurons is known to increase susceptibility to PD, and our earlier study showed a lower yield of DA-neurons in LRRK2-I1371V mutation-carrying PD patient-iPSCs. While the role of SHH in DA-neurogenesis of Floor-Plate Cells (FPCs) is known, effect of LRRK2 mutations on SHH-responsiveness of FPCs impacting DA-neuronal yield has not been studied. We investigated SHH-responsiveness of FPCs derived from LRRK2-I1371V PD patient-derived iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 expression, intracellular Ca2+ rise, and cytosolic cAMP levels upon SHH induction. Additionally, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane-fluidity and Rab8A & Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC)-FPCs overexpressing LRRK2-I1371V as well as FPCs. While total expression of Ptch1 and Smo was comparable, receptor expression on cell-surface was significantly lower in LRRK2-I1371V FPCs than in HC, with distinctly lower nuclear-expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2 I1371V exhibited a similarly reduced cell-surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared to HC upon SHH-stimulation. Both LRRK2-I1371V mutant FPCs and LRRK2-I1371V transfected SH-SY5Y and HC-FPCs further exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A & Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker Caveolin1 were also observed in them. These findings suggest that impaired SHH-responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA-neurons during ontogeny. Reduced cell-surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Autophagy favors survival of corpora lutea during the long-lasting pregnancy of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The corpus luteum (CL) is a transient endocrine gland that plays a crucial role in establishing and maintaining pregnancy. Although autophagy and apoptosis have been suggested as cooperative mechanisms, their interaction within the CL of pregnant mammals has not been thoroughly investigated. To understand the collaborative function of autophagy and apoptosis in the CL, we analyzed both mechanisms during pregnancy in the South American plains vizcacha, Lagostomus maximus. This rodent undergoes a decline in progesterone levels during mid-gestation, a reactivation of the hypothalamus-hypophysis-gonadal axis, and the incorporation of new functional secondary CL. Our analysis of autophagy markers BECLIN 1 (BECN1), SEQUESTOSOME1 (SQSTM1), Microtubule-associated protein light chain 3 (LC3B), and lysosomal-associated membrane protein 1 (LAMP1) and anti- and pro-apoptotic markers BCL2 and ACTIVE CASPASE 3 (A-C3) revealed interactive behaviors between both processes. Healthy primary and secondary CL exhibited positive expression of BECN1, SQSTM1, LC3B, and LAMP1, while regressed CL displayed enhanced expression of these autophagy markers along with nuclear A-C3. Transmission electron microscopy revealed a significant formation of autophagic vesicles in regressed CL during full-term pregnancy, whereas healthy CL exhibited a low number of autophagy vesicles. The co-localization between LC3B and SQSTM1 and LC3B with LAMP1 was observed in both healthy and regressed CL during pregnancy, while co-localization of BECN1 and BCL2 was only detected in healthy CL. LC3B and ACTIVE CASPASE 3 co-localization were detected in a subset of luteal cells within the regressing CL. We propose that autophagy could act as a survival mechanism in the CL, allowing the pregnancy to progress until full-term, while also serving as a mechanism to eliminate remnants of regressed CL, thereby providing the necessary space for subsequent follicular maturation.

Sequential glycosylations at the multibasic cleavage site of SARS-CoV-2 spike protein regulate viral activity.

The multibasic furin cleavage site at the S1/S2 boundary of the spike protein is a hallmark of SARS-CoV-2 and plays a crucial role in viral infection. However, the mechanism underlying furin activation and its regulation remain poorly understood. Here, we show that GalNAc-T3 and T7 jointly initiate clustered O-glycosylations in the furin cleavage site of the SARS-CoV-2 spike protein, which inhibit furin processing, suppress the incorporation of the spike protein into virus-like-particles and affect viral infection. Mechanistic analysis reveals that the assembly of the spike protein into virus-like particles relies on interactions between the furin-cleaved spike protein and the membrane protein of SARS-CoV-2, suggesting a possible mechanism for furin activation. Interestingly, mutations in the spike protein of the alpha and delta variants of the virus confer resistance against glycosylation by GalNAc-T3 and T7. In the omicron variant, additional mutations reverse this resistance, making the spike protein susceptible to glycosylation in vitro and sensitive to GalNAc-T3 and T7 expression in human lung cells. Our findings highlight the role of glycosylation as a defense mechanism employed by host cells against SARS-CoV-2 and shed light on the evolutionary interplay between the host and the virus.

Overexpression of PEX14 results in mistargeting to mitochondria, accompanied by organelle fragmentation and clustering in human embryonic kidney cells.

Peroxisome biogenesis disorders are caused by pathogenic variants in genes involved in biogenesis and maintenance of peroxisomes. However, mitochondria are also often affected in these diseases. Peroxisomal membrane proteins, including PEX14, have been found to mislocalise to mitochondria in cells lacking peroxisomes. Recent studies indicated that this mislocalisation contributes to mitochondrial abnormalities in PEX3-deficient patient fibroblasts cells. Here, we studied whether mitochondrial morphology is also affected in PEX3-deficient HEK293 cells and whether PEX14 mislocalises to mitochondria in these cells. Using high-resolution imaging techniques, we show that although endogenous PEX14 mislocalises to mitochondria, mitochondrial morphology was normal in PEX3-KO HEK293 cells. However, we discovered that overexpression of tagged PEX14 in wild-type HEK293 cells resulted in its mitochondrial localisation, accompanied by altered mitochondrial morphology. Our data indicate that overexpression of tagged PEX14 alone directly or indirectly cause mitochondrial abnormalities in cells containing peroxisomes.

SLC11A1 promotes kidney renal clear cell carcinoma (KIRC) progression by remodeling the tumor microenvironment.

Kidney renal clear cell carcinoma (KIRC) is a highly immune-infiltrated kidney cancer with the highest mortality rate and the greatest potential for invasion and metastasis. Solute carrier family 11 member1 (SLC11A1) is a phagosomal membrane protein located in monocytes and plays a role in innate immunity, autoimmune diseases, and infection, but its expression and biological role in KIRC is still unknown. In this study, we sought to investigate the potential value of SLC11A1 according to tumor growth and immune response in KIRC. TIMER and UALCAN database was used to analyze the expression feature and prognostic significance of SLC11A1 and its correlation with immune-related biomarkers in KIRC. Proliferation, migration, and invasion were measured using colony formation, EdU, and transwell assays. Role of SLC11A1 on KIRC tumor growth was examined by the xenograft tumor model in vivo. Effects of KIRC cells on macrophage polarization and the proliferation and apoptosis of CD8+ T cells were analyzed using flow cytometry assays. Herein, SLC11A1 was highly expressed in KIRC tissues and cell lines. SLC11A1 downregulation repressed KIRC cell proliferation, migration, invasion, macrophage, and lymphocyte immunity in vitro, as well as hindered tumor growth in vivo. SLC11A1 is significantly correlated with immune cell infiltration and immune-related biomarkers. In KIRC patients, SLC11A1 is highly expressed and positively correlated with the immune-related factors CCL2 and PD-L1. SLC11A1 induced CCL2 and PD-L1 expression, thereby activating the JAK/STAT3 pathway. SLC11A1 deficiency constrained KIRC cell malignant phenotypes and immune response via regulating CCL2 and PD-L1-mediated JAK/STAT3 pathway, providing a promising therapeutic target for KIRC treatment.

Mitochondrial Protein TAMM41 Modulates Depressive-like Behaviors.

Several lines of evidence have highlighted the crucial role of mitochondria-based therapy in depression. However, there are still less mitochondrial targets for the depression treatment. TAM41 mitochondrial translocator assembly and maintenance homolog (TAMM41) is a mitochondrial inner membrane protein for maintaining mitochondrial function, which is tightly related to many brain diseases including Alzheimer's diseases and epilepsy. Here, we investigated whether TAMM41 would be a potential target to treat depression. We found that the expression of TAMM41 was markedly lower in corticosterone-induced depression, lipopolysaccharide-induced depression, and depressed patients. Meanwhile, loss of TAMM41 resulted in increased immobility in the forced swim test (FST), tail suspension test (TST), and center time in open field test (OFT), suggesting depressive-like behaviors in mice. Moreover, genetic overexpression of TAMM41 obviously exerted antidepressant-like activities. Mechanistically, proteomics revealed that pacsin1 might be the underlying target of TAMM41. Further data supported that TAMM41 regulated the expression of pacsin1, and its antidepressant-like effect at least partially was attributed to pacsin1. In addition, exosomes containing TAMM41 was sufficient to exhibit antidepressant-like effect, suggesting an alternative strategy to exert the effect of TAMM41. Taken together, the present study demonstrates the antidepressant-like effect of TAMM41 and sheds light on its molecular mechanism. These finding provide new insights into a therapeutic strategy targeting mitochondria in the development of novel antidepressants.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Ovarian Transcriptome Analysis Reveals the Mechanism of Translucent Eggshell Formation

The presence of translucent eggshells is a type of egg quality issue that impacts egg sales. While many researchers have studied them, the exact mechanisms behind their formation remain unclear. In this study, we conducted a transcriptomic differential expression analysis of the isthmus region of the oviduct in both normal egg- and translucent egg-laying hens. The analysis revealed that differentially expressed gene pathways were predominantly concentrated in the synthesis, modification, and transport of eggshell membrane proteins, particularly collagen proteins, which provide structural support. These findings suggest that variations in the physical structure of the eggshell membrane, resulting from changes in its chemical composition, are the fundamental cause of translucent eggshell formation. This research provides a theoretical reference for reducing the occurrence of translucent eggs.