Detergent-free Method Research Articles

Engineering a Biomimetic Glomerular Filtration Barrier: Coculturing Endothelial Podocytes on Kidney ECM-Bacterial Cellulose Membrane Hybrid.

A novel avenue for advancing our understanding of kidney disease mechanisms and developing targeted therapeutics lies in overcoming the limitations of the existing in vitro models. Traditional animal models, while useful, do not fully capture the intricacies of human kidney physiology and pathophysiology. Tissue engineering offers a promising solution, yet current models often fall short in replicating the complex microarchitecture and biochemical milieu of the kidney. To address this challenge, we propose the development of a sophisticated in vitro glomerular filtration barrier (GFB) utilizing advanced biomaterials and a kidney decellularized extracellular matrix (kdECM). In our approach, we employ a bacterial cellulose membrane (BC) as a scaffold, providing a robust framework for cell growth and interaction. Coating this scaffold with kdECM hydrogel derived from caprine kidney tissue via a detergent-free decellularization method ensures the preservation of vital extracellular matrix proteins crucial for cellular compatibility and signaling. Our engineered GFB not only supports the growth of endothelial and podocyte cells but also exhibits the presence of key markers such as CD31 and nephrin, indicating successful cellular integration. Furthermore, the expression of collagen IV, an essential extracellular matrix (ECM) protein, validates the fidelity of our model in simulating cellular interactions within a kdECM matrix. Additionally, we assessed the filtration efficiency of the developed GFB model using albumin, a standard protein, to evaluate its performance under conditions that closely mimic the native physiological environment. This innovative approach, which faithfully recapitulates the native microenvironment of the glomerulus, holds immense promise for elucidating kidney disease mechanisms, conducting permeability studies, and advancing personalized therapeutic strategies. By leveraging cutting-edge biomaterials and tissue-specific coculture technology, this study can be further extended to develop GFB for the treatment of renal diseases, ultimately improving patient outcomes and quality of life.

Assessment and process optimization of high throughput biofabrication of immunocompetent breast cancer model for drug screening applications

Recent advancements in 3D cancer modeling have significantly enhanced our ability to delve into the intricacies of carcinogenesis. Despite the pharmaceutical industry’s substantial investment of both capital and time in the drug screening and development pipeline, a concerning trend persists: drug candidates screened on conventional cancer models exhibit a dismal success rate in clinical trials. One pivotal factor contributing to this discrepancy is the absence of drug testing on pathophysiologically biomimetic 3D cancer models during pre-clinical stages. Unfortunately, current manual methods of 3D cancer modeling, such as spheroids and organoids, suffer from limitations in reproducibility and scalability. In our study, we have meticulously developed 3D bioprinted breast cancer model utilizing decellularized adipose tissue-based hydrogel obtained via a detergent-free decellularization method. Our innovative printing techniques allows for rapid, high-throughput fabrication of 3D cancer models in a 96-well plate format, demonstrating unmatched scalability and reproducibility. Moreover, we have conducted extensive validation, showcasing the efficacy of our platform through drug screening assays involving two potent anti-cancer drugs, 5-Fluorouracil and PRIMA-1Met. Notably, our platform facilitates effortless imaging and gene expression analysis, streamlining the evaluation process. In a bid to enhance the relevance of our cancer model, we have introduced a heterogeneous cell population into the DAT-based bioink. Through meticulous optimization and characterization, we have successfully developed a biomimetic immunocompetent breast cancer model, complete with microenvironmental cues and diverse cell populations. This breakthrough paves the way for rapid multiplex drug screening and the development of personalized cancer models, marking a paradigm shift in cancer research and pharmaceutical development.

High Throughput Bioprinting Using Decellularized Adipose Tissue-Based Hydrogels for 3D Breast Cancer Modeling.

3D bioprinting allows rapid automated fabrication and can be applied for high throughput generation of biomimetic constructs for in vitro drug screening. Decellularized extracellular matrix (dECM) hydrogel is a popular biomaterial choice for tissue engineering and studying carcinogenesis as a tumor microenvironmental mimetic. This study proposes a method for high throughput bioprinting with decellularized adipose tissue (DAT) based hydrogels for 3D breast cancer modeling. A comparative analysis of decellularization protocol using detergent-based and detergent-free decellularization methods for caprine-origin adipose tissue is performed, and the efficacy of dECM hydrogel for 3D cancer modeling is assessed. Histological, biochemical, morphological, and biological characterization and analysis showcase the cytocompatibility of DAT hydrogel. The rheological property of DAT hydrogel and printing process optimization is assessed to select a bioprinting window to attain 3D breast cancer models. The bioprinted tissues are characterized for cellular viability and tumor cell-matrix interactions. Additionally, an approach for breast cancer modeling is shown by performing rapid high throughput bioprinting in a 96-well plate format, and in vitro drug screening using 5-fluorouracil is performed on 3D bioprinted microtumors. The results of this study suggest that high throughput bioprinting of cancer models can potentially have downstream clinical applications like multi-drug screening platforms and personalized disease models.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

Preparation of Corneal Tissue Matrix Bioink for 3D Bioprinting.

There are many protocols available to decellularize tissues for the preparation of bioink for 3D bioprinting purposes. Almost all the methods comprise multiple chemicals and enzymes in different combinations. Here we describe the usage of sodium chloride that enables the decellularization of corneal tissues from human and animal sources, which is a simple, rapid, and detergent-free method, unlike conventional decellularization protocols. The method described here is for cornea tissue decellularization and its digestion and bioink preparation for 3D bioprinting applications. We demonstrate the efficient decellularization of tissues by retaining the extracellular matrix.

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening.

Membrane protein (MP)-based biomaterials have a wide range of applications in drug screening, antigen detection, and ligand-receptor interaction analysis. Traditional MP immobilization methods have the disadvantage of disordered protein immobilization orientation, leading to the shielded binding domain and unreliable binding pattern. Herein, we describe a site-specific covalent immobilization of MPs, which utilizes the styrene maleic acid (SMA) detergent-free extraction method of MPs as well as the covalent reaction between His-tag and divinyl sulfone (DVS). As an example, we covalently immobilized angiotensin-converting enzyme 2 (ACE2) on a cell membrane chromatography system (ACE2-His-SMALPs/CMC) in a site-specific manner and verified the specificity and stability of this system. This technique significantly improves the service life compared to the physisorption CMC column. The improved protein immobilization strategies of the ACE2-His-SMALPs/CMC system enable it to effectively recognize SARS-CoV-2 pseudoviral particles as well as detect viral particles in ambient air once combined with an aerosol collector; as a powerful ligand biosensor, the ACE2-His-SMALPs/CMC system was used to screen for compounds with anti-SARS-CoV-2 pseudovirus activity. In conclusion, the optimized MP immobilization strategy has been successfully applied to CMC technology, showing enhanced stability and sensitivity, which can provide an efficient and convenient membrane protein immobilization method for biomaterials.

Decellularized bladder as scaffold to support proliferation and functionality of insulin-secreting pancreatic cells.

Loss in the number or function of insulin-producing β-cells in pancreatic islets has been associated with diabetes mellitus. Although islet transplantation can be an alternative treatment, complications such as apoptosis, ischaemia and loss of viability have been reported. The use of decellularized organs as scaffolds in tissue engineering is of interest owing to the unique ultrastructure and composition of the extracellular matrix (ECM) believed to act on tissue regeneration. In this study, a cell culture system has been designed to study the effect of decellularized porcine bladder pieces on INS-1 cells, a cell line secreting insulin in response to glucose stimulation. Porcine bladders were decellularized using two techniques: a detergent-containing and a detergent-free methods. The resulting ECMs were characterized for the removal of both cells and dsDNA. INS-1 cells were not viable on ECM produced using detergent (i.e., sodium dodecyl sulfate). INS-1 cells were visualized following 7 days of culture on detergent-free decellularized bladders using a cell viability and metabolism assay (MTT) and cell proliferation quantified (CyQUANT™ NF Cell Proliferation Assay). Further, glucose-stimulated insulin secretion and immunostaining confirmed that cells were functional in response to glucose stimulation, as well as they expressed insulin and interacted with the detergent-free produced ECM, respectively.

A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community.

A New Model of Esophageal Cancers by Using a Detergent-Free Decellularized Matrix in a Perfusion Bioreactor.

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Detergent-Free Decellularization Preserves the Mechanical and Biological Integrity of Murine Tendon.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. In this study, we adapted and established the advantages of a detergent-free decellularization method that relies on latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/"provisional"), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.

Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research.

Decellularized extracellular matrices (ECMs) are being extensively used for tissue engineering purposes and detergents are predominantly used for this. A facile detergent-free decellularization method is developed for dermal matrix and compared it with the most used detergent-based decellularization methods. An optimized, single-step, cost-effective Hypotonic/Hypertonic (H/H) Sodium Chloride (NaCl) solutions-based method is employed to decellularize goat skin that resulted in much higher yield than other methods. The ECM composition, mechanical property, and cytocompatibility are evaluated and compared with other decellularization methods. Furthermore, this H/H-treated decellularized dermal ECM (ddECM) exhibits a residual DNA content of <50ngmg-1 of dry tissue. Moreover, 85.64±3.01% of glycosaminoglycans and 65.53±2.9% collagen are retained compared to the native tissue, which is higher than the ddECMs prepared by other methods. The cellular response is superior in ddECM (H/H) than other ddECMs prepared by detergent-based methods. Additionally, a bioink is formulated with the ddECM (H/H), showing good shear thinning and shear recovery properties. Process optimization in terms of print speed, flow rate, and viscosity is done to obtain a bioprinting window for ddECM bioink. The printed constructs with optimized parameters have adequate mechanical and cell adhesive properties and excellent isotropic cellular alignment.

Decellularization of human amniotic membrane using detergent-free methods: Possibilities in tissue engineering

The human amniotic membrane (HAM) is widely used as a natural scaffold in tissue engineering due to its excellent biological characteristics, including anti-microbial, anti-inflammatory, low immunogenicity, and pro-angiogenic properties. This study aimed to develop simple and cost-effective protocols for the decellularization of HAM (d-HAM) using detergent-free methods, i.e., mechanical force (brushing) and physical treatment (heating 45–55 °C). The effectiveness of the methods of interest was compared with a chemical-based approach (EDTA + NaOH + NH4Cl). The prepared d-HAMs were characterized using a series of physico-chemical, mechanical, and biological evaluations. The results from DAPI staining revealed that the chemical method could completely remove epithelial cells from HAM, while the two other approaches only reduced the number of epithelial cells. All three decellularization methods led to a sharp reduction (P < 0.001) in the DNA content of the tissue samples (< 50 ng/mg). Histological evaluations showed the preservation of the d-HAMs’ integrity along with the conservation of collagen and glycosaminoglycans (GAGs). Although the chemical method caused the lowest mechanical deterioration (3.55 MPa in ultimate tensile stress), the mechanical method preserved the highest hydroxyproline levels (3.13 mg/mL). On the other hand, the physical method (heating to 45 and 50 °C) encouraged cell proliferation more than the chemical and mechanical approaches. All of the samples proved to be suitable for cell attachment and could induce cell migration. In conclusion, the present study showed that the use of detergent-free protocols is applicable for the decellularization of HAM, and the obtained tissues may be considered as inexpensive dressings for numerous tissue engineering applications.

FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.

Proteomic and Bioinformatic Analysis of Decellularized Pancreatic Extracellular Matrices.

Tissue microenvironments are rich in signaling molecules. However, factors in the tissue matrix that can serve as tissue-specific cues for engineering pancreatic tissues have not been thoroughly identified. In this study, we performed a comprehensive proteomic analysis of porcine decellularized pancreatic extracellular matrix (dpECM). By profiling dpECM collected from subjects of different ages and genders, we showed that the detergent-free decellularization method developed in this study permits the preservation of approximately 62.4% more proteins than a detergent-based method. In addition, we demonstrated that dpECM prepared from young pigs contained approximately 68.5% more extracellular matrix proteins than those prepared from adult pigs. Furthermore, we categorized dpECM proteins by biological process, molecular function, and cellular component through gene ontology analysis. Our study results also suggested that the protein composition of dpECM is significantly different between male and female animals while a KEGG enrichment pathway analysis revealed that dpECM protein profiling varies significantly depending on age. This study provides the proteome of pancreatic decellularized ECM in different animal ages and genders, which will help identify the bioactive molecules that are pivotal in creating tissue-specific cues for engineering tissues in vitro.

Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass spectrometry.

Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram-negative bacteria and apply to clinical mass spectrometry. The Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC-MS/MS and MALDI-TOF MS. The effectiveness of the OS lysis method for KPC-2-producing Enterobacteriaceae clinical isolates were then confirmed by MALDI-TOF MS. The optimized OS lysis using KPC-2 producing E. coli standard cells showed a high yield of KPC-2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC-2 protein significantly increased in MALDI-TOF MS analysis. Nineteen clinical isolates were validated by MALDI-TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n-octyl-β-D-glucopyranoside) (p<0.001). This study provides a straightforward, rapid, affordable, and detergent-free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS-based clinical diagnostics.

Detergent-free systems for structural studies of membrane proteins.

Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide accurate structural information to understand their mechanisms of action. The advance of membrane protein structural biology has primarily relied on detergent-based methods over the past several decades. However, detergent-based approaches have significant drawbacks because detergents often damage the native protein-lipid interactions, which are often crucial for maintaining the natural structure and function of membrane proteins. Detergent-free methods recently have emerged as alternatives with a great promise, e.g. for high-resolution structure determinations of membrane proteins in their native cell membrane lipid environments. This minireview critically examines the current status of detergent-free methods by a comparative analysis of five groups of membrane protein structures determined using detergent-free and detergent-based methods. This analysis reveals that current detergent-free systems, such as the styrene-maleic acid lipid particles (SMALP), the diisobutyl maleic acid lipid particles (DIBMALP), and the cycloalkane-modified amphiphile polymer (CyclAPol) technologies are not better than detergent-based approaches in terms of maintenance of native cell membrane lipids on the transmembrane domain and high-resolution structure determination. However, another detergent-free technology, the native cell membrane nanoparticles (NCMN) system, demonstrated improved maintenance of native cell membrane lipids with the studied membrane proteins, and produced particles that were suitable for high-resolution structural analysis. The ongoing development of new membrane-active polymers and their optimization will facilitate the maturation of these new detergent-free systems.

Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.

A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome

The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.

Non-detergent Isolation of Membrane Structures from Beet Plasmalemma and Tonoplast Having Lipid Composition Characteristic of Rafts.

Vacuolar and plasma membranes were isolated by a detergent-free method from beet roots (Beta vulgaris L.), and were fractionated in a sucrose density gradient of 15-60% by high-speed centrifugation at 200,000×g during 18h. The membrane material distributed over the sucrose density gradient was analyzed for the presence of lipids characteristic of raft structures in different zones of the gradient. The quantitative and qualitative content of lipids and sterols, and the composition of fatty acids were analyzed. Some membrane structures differing in their biochemical characteristics were revealed to be located in different zones of the sucrose gradient. The results of the analysis allowed us to identify three zones in the sucrose gradient after the vacuolar membrane fractionation and two zones in the plasma membrane where membrane structures, which may be defined as rafts for their lipid composition, were presented.