Membrane Proteins/metabolism* ; Protein Binding

Hemoglobinuria, Paroxysmal ; metabolism ; Humans ; Membrane Proteins ; biosynthesis

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.
Carrier Proteins ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Interferometry ; Membrane Proteins/metabolism*

OBJECTIVETo explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc.

METHODSThe expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method.

RESULTS(1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time.

CONCLUSIONThe membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.

Cell Line ; Cell Membrane ; genetics ; metabolism ; Endocytosis ; Humans ; Membrane Proteins ; genetics ; metabolism ; PrPC Proteins ; genetics ; metabolism ; Protein Binding ; Protein Transport

A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(epsilon)-(gamma-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of omega-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.
Animal ; Cell Membrane/metabolism ; Epidermis/metabolism* ; Epidermis/chemistry* ; Human ; Ichthyosis/metabolism ; Ichthyosis/genetics ; Keratinocytes/metabolism* ; Keratinocytes/chemistry ; Membrane Lipids/metabolism* ; Membrane Proteins/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism

Animals ; Cell Membrane ; chemistry ; metabolism ; Humans ; Membrane Proteins ; chemistry ; metabolism ; Protein Structure, Secondary

While the field of ATP synthase research has a long history filled with landmark discoveries, recent structural works provide us with important insights into the mechanisms that links the proton movement with the rotation of the Fo motor. Here, we propose a mechanism of unidirectional rotation of the Fo complex, which is in agreement with these new structural insights as well as our more general ΔΨ-driving hypothesis of membrane proteins: A proton path in the rotor-stator interface is formed dynamically in concert with the rotation of the Fo rotor. The trajectory of the proton viewed in the reference system of the rotor (R-path) must lag behind that of the stator (S-path). The proton moves from a higher energy site to a lower site following both trajectories simultaneously. The two trajectories meet each other at the transient proton-binding site, resulting in a relative rotation between the rotor and stator. The kinetic energy of protons gained from ΔΨ is transferred to the c-ring as the protons are captured sequentially by the binding sites along the proton path, thus driving the unidirectional rotation of the c-ring. Our ΔΨ-driving hypothesis on Fo motor is an attempt to unveil the robust mechanism of energy conversion in the highly conserved, ubiquitously expressed rotary ATP synthases.
Membrane Potentials ; physiology ; Membrane Proteins ; chemistry ; metabolism ; Mitochondrial Proton-Translocating ATPases ; chemistry ; metabolism ; Protein Conformation

Anthocyanin biosynthesis is one of the thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanins into the vacuole. A clear picture of the dynamic trafficking of flavonoids is only now beginning to emerge. So far four different models have been proposed to explain the transport of anthocyanins from biosynthetic sites to the central vacuole, and four types of transporters have been found associated with the transport of anthocyanins: glutathione S-transferase, multidrug resistance-associated protein, multidrug and toxic compound extrusion, bilitranslocase-homologue. The functions of these proteins and related genes have also been studied. Although different models have been proposed, cellular and subcellular information is still lacking for reconciliation of different lines of evidence in various anthocyanin sequestration studies. According to the information available, through sequence analysis, gene expression analysis, subcellular positioning and complementation experiments, the function and location of these transporters can be explored, and the anthocyanin transport mechanism can be better understood.
Anthocyanins ; metabolism ; Biological Transport ; Glutathione Transferase ; metabolism ; Membrane Transport Proteins ; metabolism ; Multidrug Resistance-Associated Proteins ; metabolism ; Plants ; metabolism ; Vacuoles ; metabolism

As a functional membrane protein, drug transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The functional omission or inhibition of drug transporters is believed to be involved in the drug-drug interaction and pathogenesis of certain diseases. Understanding the function of drug transporters is highly significant in terms of pharmacokinetics, pharmacodynamics and toxicity of drugs. This article summarized the methods for the study of drug transporters in vitro and in vivo.
Biological Transport ; Drug Interactions ; Humans ; Membrane Transport Proteins ; metabolism

Rafts, cholesterol- and sphingolipid-rich membrane microdomains, have been shown to play an important role in immune cell activation. More recently rafts were implicated in the signal transduction by members of the TNF receptor (TNFR) family. In this study, we provide evidences that the raft microdomain has a crucial role in RANK (receptor activator of NF-kappaB) signaling. We found that the majority of the ectopically expressed RANK and substantial portion of endogenous TRAF2 and TRAF6 were detected in the low-density raft fractions. In addition, TRAF6 association with rafts was increased by RANKL stimulation. The disruption of rafts blocked the TRAF6 translocation by RANK ligand and impeded the interaction between RANK and TRAF6. Our observations demonstrate that proper RANK signaling requires the function of raft membrane microdomains.
Carrier Proteins/metabolism ; Glycoproteins/*metabolism ; Human ; Membrane Glycoproteins/metabolism ; Membrane Microdomains/*metabolism ; Protein Transport/physiology ; Proteins/*metabolism ; Receptors, Cytoplasmic and Nuclear/*metabolism