Membrane protein reconstitution for functional and structural studies

Wang, LiGuo G.; Tonggu, Lige

doi:10.1007/s11427-014-4769-0

Cited by 27 publications

(23 citation statements)

References 70 publications

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“…Proteoliposomes based on large unilamellar vesicles (LUVs, from 50 to 400 nm) are promising systems for drug delivery, mainly owing to their size, hydrophobic/hydrophilic properties, biocompatibility, biodegradability and low toxicity in the human body [17–21]. During the last decade, these biomimetic systems have gained interest as tools for biophysical studies of lipid–protein interactions as well as for biotechnological applications [16,22]. There is an increasing interest in the study and characterization of the structure, geometry, size and physical properties of proteoliposomes prepared for medical applications.…”

Section: Introductionmentioning

confidence: 99%

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Bolean

Borin

Simão

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes’ membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.

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Section: Introductionmentioning

confidence: 99%

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Bolean

Borin

Simão

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…ClC-7 serves to transport Cl − across lysosomal membranes (Kasper et al, 2005; Lange et al, 2006; Graves et al, 2008). More recently, the large conductance Ca 2+ -activated K + (BK) channel, previously only known to work on the plasma membrane and mitochondria (Xu et al, 2002; Wang and Tonggu, 2015), was found to reside and function on endolysosomal membranes of several different cell types, especially nonexcitable cells that were not known to possess functional BK channels before (Cao et al, 2015b). Remarkably, BK is unlikely the only K + channel type present on these acidic organelles.…”

Section: Introductionmentioning

confidence: 99%

Regulation of lysosomal ion homeostasis by channels and transporters

Xiong

Zhu

2016

Sci. China Life Sci.

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Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H+, Ca2+, Na+, K+, and Cl− across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

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“…Membrane proteins are involved in various critical biological processes [65], which are believed to mediate attachment and invasion of Cryptosporidium . In the present study, our first identification of CpEno on the surface by immunofluorescence examinations indicates that this protein may be a membrane-related protein.…”

Section: Discussionmentioning

confidence: 99%

Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

Yang²,

Huang³

et al. 2017

Parasites Vectors

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BackgroundEnolase is an essential multifunctional glycolytic enzyme that is involved in many biological processes of apicomplexan protozoa, such as adhesion and invasion. However, the characteristics of enolase in Cryptosporidium parvum, including the location on the oocyst and the enzyme activity, remain unclear.MethodsThe C. parvum enolase gene (cpeno) was amplified by RT-PCR and sequenced. The deduced amino acid sequence was analysed by bioinformatics software. The gene was expressed in Escherichia coli BL21 (DE3) and purified recombinant protein was used for enzyme activity analysis, binding experiments and antibody preparation. The localisation of enolase on oocysts was examined via immunofluorescence techniques.ResultsA 1,350 bp DNA sequence was amplified from cDNA taken from C. parvum oocysts. The deduced amino acids sequence of C. parvum enolase (CpEno) had 82.1% homology with Cryptosporidium muris enolase, and 54.7–68.0% homology with others selected species. Western blot analysis indicated that recombinant C. parvum enolase (rCpEno) could be recognised by C. parvum-infected cattle sera. Immunolocalization testing showed that CpEno was found to locate mainly on the surface of oocysts. The enzyme activity was 33.5 U/mg, and the Michaelis constant (K m) was 0.571 mM/l. Kinetic measurements revealed that the most suitable pH value was 7.0–7.5, and there were only minor effects on the activity of rCpEno with a change in the reaction temperature. The enzyme activity decreased when the Ca2+, K+, Mg2+ and Na+ concentrations of the reaction solution increased. The binding assays demonstrated that rCpEno could bind to human plasminogen.ConclusionThis study is the first report of immunolocation, binding activity and enzyme characteristics of CpEno. The results of this study suggest that the surface-associated CpEno not only functions as a glycolytic enzyme but may also participate in attachment and invasion process of the parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2200-y) contains supplementary material, which is available to authorized users.

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Membrane protein reconstitution for functional and structural studies

Cited by 27 publications

References 70 publications

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Topographic analysis by atomic force microscopy of proteoliposomes matrix vesicle mimetics harboring TNAP and AnxA5

Regulation of lysosomal ion homeostasis by channels and transporters

Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

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