scholarly journals Transmembrane topology and oligomeric nature of an astrocytic membrane protein, MLC1

Open Biology ◽  
2021 ◽  
Vol 11 (12) ◽  
Author(s):  
Junmo Hwang ◽  
Kunwoong Park ◽  
Ga-Young Lee ◽  
Bo Young Yoon ◽  
Hyunmin Kim ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Protein Complexes ◽  
Functional Characterization ◽  
Biochemical Properties ◽  
Genetic Mutations ◽  
Transmembrane Topology ◽  
Trimeric Complex ◽  
C Terminus

MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

scholarly journals Functional characterization of three flavonoid glycosyltransferases from Andrographis paniculata

2019 ◽  
Vol 6 (6) ◽  
pp. 190150 ◽  
Author(s):  
Yuan Li ◽  
Xin-Lin Li ◽  
Chang-Jiang-Sheng Lai ◽  
Rui-Shan Wang ◽  
Li-Ping Kang ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Biochemical Properties ◽  
Flavonoid Glycoside ◽  
Andrographis Paniculata ◽  
Biologically Active ◽  
Flavonoid Glycosides ◽  
Asian Countries ◽  
Pharmacological Activities ◽  
Different Types

Andrographis paniculata is an important traditional medicinal herb in South and Southeast Asian countries with diverse pharmacological activities that contains various flavonoids and flavonoid glycosides. Glycosylation can transform aglycones into more stable, biologically active and structurally diverse glycosides. Here, we report three glycosyltransferases from the leaves of A. paniculata (ApUFGTs) that presented wide substrate spectra for flavonoid glycosylation and exhibited multi-site glycosylation on the substrate molecules. They acted on the 7-OH position of the A ring and were able to glycosylate several other different types of compounds. The biochemical properties and phylogenetic analysis of these glycosyltransferases were also investigated. This study provides a basis for further research on the cloning of genes involved in glycosylation from A. paniculata and offers opportunities for enhancing flavonoid glycoside production in heterologous hosts. These enzymes are expected to become effective tools for drug discovery and for the biosynthesis of derivatives via flavonoid glycosylation.

scholarly journals High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes ofDesulfovibrio vulgaris

2012 ◽  
Vol 11 (12) ◽  
pp. 5720-5735 ◽  
Author(s):  
Peter J. Walian ◽  
Simon Allen ◽  
Maxim Shatsky ◽  
Lucy Zeng ◽  
Evelin D. Szakal ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
High Throughput ◽  
Protein Complexes ◽  
Isolation And Characterization ◽  
Membrane Protein Complexes

scholarly journals Proteomics Strategy for Identifying Candidate Bioactive Proteins in Complex Mixtures: Application to the Platelet Releasate

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Roisin O'Connor ◽  
Lorna M. Cryan ◽  
Kieran Wynne ◽  
Andreas de Stefani ◽  
Desmond Fitzgerald ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Functional Characterization ◽  
Biological Tissues ◽  
Biochemical Properties ◽  
Human Platelets ◽  
Intact Proteins ◽  
Large Numbers ◽  
Wide Range ◽  
Platelet Releasate

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

scholarly journals Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2425-2440 ◽  
Author(s):  
Cunle Wu ◽  
Ekkehard Leberer ◽  
David Y. Thomas ◽  
Malcolm Whiteway
Keyword(s):  
Protein Kinase ◽  
Functional Characterization ◽  
Hog Pathway ◽  
C Terminus ◽  
Extracellular Signal ◽  
Osmotic Stress Response ◽  
N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Characterization of Rab3A, Rab3B and Rab3C: different biochemical properties and intracellular localization in bovine chromaffin cells

1997 ◽  
Vol 324 (1) ◽  
pp. 85-90 ◽  
Author(s):  
Chong-Gee LIN ◽  
Yu-Chi LIN ◽  
Hwan-Wun LIU ◽  
Lung-Sen KAO
Keyword(s):  
Subcellular Localization ◽  
Binding Affinity ◽  
Chromaffin Cells ◽  
Intracellular Localization ◽  
Biochemical Properties ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Bovine Chromaffin Cells ◽  
Adrenal Chromaffin

In this study we examined the biochemical properties and subcellular localization of Rab3A, Rab3B and Rab3C in bovine adrenal chromaffin cells. The Kd for guanosine 5′-[γ-thio]triphosphate (GTP[S]) of the three Rab3 proteins was 15, 2700 and 204 nM for Rab3A, Rab3B and Rab3C respectively. The intrinsic GTPase activity of the three Rab3 proteins seemed similar and was increased approx. 3-fold by bovine chromaffin cell lysate. Truncation of the C-terminal 31 amino acid residues decreased the binding affinity for GTP[S] of the three Rab3 proteins. When the C-terminus of Rab3C was replaced with that of Rab3A, the binding affinity of Rab3C for GTP[S] was decreased, but the replacement did not affect the affinity of Rab3B for GTP[S]. Immunostaining experiments showed that Rab3A, Rab3B and Rab3C are localized separately within chromaffin cells. Anti-Rab3A and anti-Rab3C antibodies stained vesicle-like structures, whereas anti-Rab3B antibody distinctly stained the plasma membrane. In summary, bovine chromaffin cells express the three Rab3 proteins but the subcellular localization and biochemical properties of the three Rab3 proteins are distinct.

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