scholarly journals Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

2009 ◽  
Vol 418 (3) ◽  
pp. 651-664 ◽  
Author(s):  
Stefano Del Duca ◽  
Donatella Serafini-Fracassini ◽  
Philip Bonner ◽  
Mauro Cresti ◽  
Giampiero Cai
Keyword(s):  
Molecular Mass ◽  
Functional Properties ◽  
Actin Filaments ◽  
Hydrophobic Interaction Chromatography ◽  
High Molecular Mass ◽  
Acyl Acceptor ◽  
Post Translational Modifications ◽  
Calcium Dependent ◽  
Cross Links ◽  
Guinea Pig Liver

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein–cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

Characterization and functional properties of soybean high-molecular-mass polysaccharide complex

2012 ◽  
Vol 29 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Akihiro Nakamura ◽  
Nanae Fujii ◽  
Junko Tobe ◽  
Norifumi Adachi ◽  
Motohiko Hirotsuka
Keyword(s):  
Molecular Mass ◽  
Functional Properties ◽  
High Molecular Mass ◽  
Polysaccharide Complex

scholarly journals Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex

2003 ◽  
Vol 370 (2) ◽  
pp. 469-477 ◽  
Author(s):  
Katherine M. HANNAN ◽  
George THOMAS ◽  
Richard B. PEARSON
Keyword(s):  
Ribosomal Protein ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Phosphorylation Sites ◽  
S6 Kinase ◽  
Calcium Dependent ◽  
Ribosomal Protein S6 ◽  
Calcium Chelation ◽  
Regulatory Phosphorylation ◽  
Ribosomal Protein S6 Kinase

The mitogen-stimulated protein kinase p70 ribosomal protein S6 kinase 1 (S6K1) is a key enzyme in the regulation of cell growth and proliferation. Activation of S6K1 requires a complex, ordered series of conformational changes and phosphorylation reactions. While the role of sequential, multi-site phosphorylation has been extensively detailed, characterization of the priming step required to initiate this cascade has remained elusive. In the present study we show for the first time that this priming process is dependent on calcium. Calcium-dependent regulation of S6K1 did not specifically target Thr-229 and Thr-389, the key regulatory phosphorylation sites; rather, calcium chelation resulted in a global inhibition of S6K1 phosphorylation. Mutation of individual phosphorylation sites in the auto-inhibitory and hydrophobic domains to acidic residues (to mimic phosphorylation) yields a kinase that remains sensitive to calcium chelation, while the combined mutations alleviate the requirement for calcium. Furthermore, deletion of the C-terminal residues (398—502) also renders the kinase insensitive to calcium. We hypothesize that the initial calcium-dependent process is required to release an inhibitory interaction between the C- and N-termini of S6K1, thus allowing phosphorylation of these key domains. The requirement for this priming step can only be overcome by mutations mimicking the phosphorylation of both the auto-inhibitory and hydrophobic domains. We further propose that the priming event involves formation of a calcium-dependent protein complex that releases the interaction between the N- and C-termini. S6K1 is then accessible for activation by the kinases that target the known regulatory phosphorylation sites. Consistent with this hypothesis, serum stimulation of S6K1 activity is associated with its incorporation into a calcium-dependent high-molecular-mass complex.

Purification and Characterization of a Novel Metalloproteinase, Acurhagin, from Agkistrodon acutus Venom

2002 ◽  
Vol 87 (04) ◽  
pp. 641-650 ◽  
Author(s):  
Wen-Jeng Wang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Molecular Mass ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Crude Venom ◽  
Reducing Conditions ◽  
Sds Page

SummaryAcurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anionexchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aα. chain of fibrinogen, followed by Bβ chain, while γ chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37° C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrinlike and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of plateletrich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

A high molecular mass non-muscle tropomyosin isoform stimulates retrograde organelle transport

1996 ◽  
Vol 109 (5) ◽  
pp. 981-989 ◽  
Author(s):  
R.J. Pelham ◽  
J.J. Lin ◽  
Y.L. Wang
Keyword(s):  
Molecular Mass ◽  
Actin Filaments ◽  
Rat Kidney ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
High Molecular Mass ◽  
Organelle Transport ◽  
Cellular Functions ◽  
Myosin I ◽  
Perinuclear Area

Although non-muscle tropomyosins (TM) have been implicated in various cellular functions, such as stabilization of actin filaments and possibly regulation of organelle transport, their physiological role is still poorly understood. We have probed the role of a high molecular mass isoform of human fibroblast TM, hTM3, in regulating organelle transport by microinjecting an excess amount of bacterially-expressed protein into normal rat kidney (NRK) epithelial cells. The microinjection induced the dramatic retrograde translocation of organelles into the perinuclear area. Microinjection of hTM5, a low molecular mass isoform had no effect on organelle distribution. Fluorescent staining indicated that hTM3 injection stimulated the retrograde movement of both mitochondria and lysosomes. Moreover, both myosin I and cytoplasmic dynein were found to redistribute with the translocated organelles to the perinuclear area, indicating that these organelles were able to move along both microtubules and actin filaments. The involvement of microtubules was further suggested by the partial inhibition of hTM3-induced organelle movement by the microtubule-depolymerizing drug nocodazole. Our results, along with previous genetic and antibody microinjection studies, suggest that hTM3 may be involved in the regulation of organelle transport.

High molecular mass kininogen inhibits cathepsin G-induced platelet activation by forming a complex with cathepsin G

2001 ◽  
Vol 2 (6) ◽  
pp. 371-377 ◽  
Author(s):  
Tarek E Selim ◽  
Hayam R Ghoneim ◽  
Hassan A Abdel Ghaffar ◽  
Robert W Colman ◽  
Raul A Dela Cadena
Keyword(s):  
Molecular Mass ◽  
Platelet Activation ◽  
High Molecular Mass ◽  
Cathepsin G

scholarly journals Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 453
Author(s):  
Sebastian Estrada-Gómez ◽  
Leidy Johana Vargas-Muñoz ◽  
Cesar Segura Latorre ◽  
Monica Maria Saldarriaga-Cordoba ◽  
Claudia Marcela Arenas-Gómez
Keyword(s):  
Enzymatic Activity ◽  
Molecular Mass ◽  
Evolutionary Relationship ◽  
Venom Gland ◽  
Duplication Event ◽  
High Molecular Mass ◽  
Amino Acid Sequences ◽  
Secretory Proteins ◽  
Phospholipases A2 ◽  
Kunitz Type

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

scholarly journals Age-dependent ataxia and neurodegeneration caused by an αII spectrin mutation with impaired regulation of its calpain sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Arkadiusz Miazek ◽  
Michał Zalas ◽  
Joanna Skrzymowska ◽  
Bryan A. Bogin ◽  
Krzysztof Grzymajło ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Neuronal Membrane ◽  
Post Translational Modifications ◽  
Calcium Dependent ◽  
Enhanced Sensitivity ◽  
Embryonic Lethal ◽  
Progressive Ataxia ◽  
Substrate Level ◽  
Age Dependent ◽  
Excessive Activation

AbstractThe neuronal membrane-associated periodic spectrin skeleton (MPS) contributes to neuronal development, remodeling, and organization. Post-translational modifications impinge on spectrin, the major component of the MPS, but their role remains poorly understood. One modification targeting spectrin is cleavage by calpains, a family of calcium-activated proteases. Spectrin cleavage is regulated by activated calpain, but also by the calcium-dependent binding of calmodulin (CaM) to spectrin. The physiologic significance of this balance between calpain activation and substrate-level regulation of spectrin cleavage is unknown. We report a strain of C57BL/6J mice harboring a single αII spectrin point mutation (Sptan1 c.3293G > A:p.R1098Q) with reduced CaM affinity and intrinsically enhanced sensitivity to calpain proteolysis. Homozygotes are embryonic lethal. Newborn heterozygotes of either gender appear normal, but soon develop a progressive ataxia characterized biochemically by accelerated calpain-mediated spectrin cleavage and morphologically by disruption of axonal and dendritic integrity and global neurodegeneration. Molecular modeling predicts unconstrained exposure of the mutant spectrin’s calpain-cleavage site. These results reveal the critical importance of substrate-level regulation of spectrin cleavage for the maintenance of neuronal integrity. Given that excessive activation of calpain proteases is a common feature of neurodegenerative disease and traumatic encephalopathy, we propose that damage to the spectrin MPS may contribute to the neuropathology of many disorders.

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