scholarly journals The Quaternary Structure of Bovine alpha-Crystallin. Surface Probing by Limited Proteolysis in vitro

1979 ◽  
Vol 96 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Roland J. SIEZEN ◽  
Herman J. HOENDERS
Keyword(s):  
Quaternary Structure ◽  
Limited Proteolysis ◽  
Alpha Crystallin

scholarly journals An autonomous, but INSIG-modulated, role for the Sterol Sensing Domain in mallostery-regulated ERAD of yeast HMG-CoA reductase

2020 ◽  
pp. jbc.RA120.015910
Author(s):  
Margaret A Wangeline ◽  
Randolph Y Hampton
Keyword(s):  
Binding Site ◽  
Quaternary Structure ◽  
Limited Proteolysis ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Level Analysis

HMG-CoA reductase (HMGR) undergoes feedback-regulated degradation as part of sterol pathway control. Degradation of the yeast HMGR isozyme Hmg2 is controlled by the sterol pathway intermediate GGPP, which causes misfolding of Hmg2, leading to degradation by the HRD pathway; we call this process mallostery. We evaluated the role of the Hmg2 sterol sensing domain (SSD) in mallostery, as well as the involvement of the highly conserved INSIG proteins. We show that the Hmg2 SSD is critical for regulated degradation of Hmg2 and required for mallosteric misfolding of GGPP as studied by in vitro limited proteolysis. The Hmg2 SSD functions independently of conserved yeast INSIG proteins, but its function was modulated by INSIG, thus imposing a second layer of control on Hmg2 regulation. Mutant analyses indicated that SSD-mediated mallostery occurred prior to and independent of HRD-dependent ubiquitination. GGPP-dependent misfolding was still extant but occurred at a much slower rate in the absence of a functional SSD, indicating that the SSD facilitates a physiologically useful rate of GGPP response, and implying that the SSD is not a binding site for GGPP. Non-functional SSD mutants allowed us to test the importance of Hmg2 quaternary structure in mallostery:  a non-responsive Hmg2 SSD mutant strongly suppressed regulation of a co-expressed, normal Hmg2. Finally, we have found that GGPP-regulated misfolding occurred in detergent-solubilized Hmg2, a feature that will allow next-level analysis of the mechanism of this novel tactic of ligand-regulated misfolding.

scholarly journals An autonomous, but INSIG-modulated, role for the Sterol Sensing Domain in mallostery-regulated ERAD of yeast HMG-CoA reductase

2020 ◽  
Author(s):  
Margaret A Wangeline ◽  
Randolph Y Hampton
Keyword(s):  
Protein Quality ◽  
Quaternary Structure ◽  
Limited Proteolysis ◽  
Hmg Coa Reductase ◽  
Autonomous Action ◽  
Pathway Regulation ◽  
Coa Reductase ◽  
Related Proteins

AbstractHMG-CoA reductase (HMGR) undergoes feedback regulated degradation as part of sterol pathway control. Degradation of the yeast HMGR isozyme Hmg2 is controlled by the sterol pathway intermediate GGPP, which causes misfolding of Hmg2 to enhance its ERAD by the HRD pathway. GGPP-dependent reversible misfolding of Hmg2 is remarkably similar to classic allosteric control; we recently labeled this process mallostery to fuse the ideas of misfolding and allostery. We have evaluated the role of the Hmg2 sterol sensing domain (SSD) in mallostery, and the involvement of highly conserved INSIG proteins in SSD function. The SSD is a membrane-embedded motif found in many sterol-related proteins. The Hmg2 SSD was critical for in vivo regulated degradation of Hmg2, and required for mallosteric misfolding of GGPP as studied by in vitro limited proteolysis. The Hmg2 SSD functions in mallostery independently of conserved yeast INSIG proteins. However, this autonomous action of the SSD was modulated by INSIG, thus imposing a second layer of control on Hmg2 regulation. SSD-mediated mallostery occurs prior to HRD dependent ubiquitination, defining a pathway regulation involving SSD-mediated misfolding followed by HRD dependent ubiquitination. GGPP dependent misfolding occurred at a much slower rate in the absence of a functional SSD, indicating that the SSD functions to allow physiologically useful rate of GGPP response, and implying that the SSD is not a binding site for GGPP. We used unresponsive Hmg2 SSD mutants to test the importance of quaternary structure in mallosteric regulation: the presence of a non-responsive Hmg2 mutant strongly suppressed regulation of a co-expressed, normal Hmg2. Finally, we have found that GGPP regulated misfolding occurred in detergent solubilized Hmg2, indicating that the mallosteric response is an intrinsic feature of the Hmg2 multimer. The preserved response of Hmg2 when in micellar solution will allow next-level studies on the structural and biophysical features of this novel fusion of regulation and protein quality control.

scholarly journals alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract.

1993 ◽  
Vol 268 (25) ◽  
pp. 18844-18849 ◽  
Author(s):  
M.J. Kelley ◽  
L.L. David ◽  
N. Iwasaki ◽  
J. Wright ◽  
T.R. Shearer
Keyword(s):  
Chaperone Activity ◽  
Alpha Crystallin ◽  
Selenite Cataract ◽  
Calpain Ii

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins

1983 ◽  
Vol 3 (6) ◽  
pp. 1146-1156
Author(s):  
W J Nelson ◽  
P Traub
Keyword(s):  
Intermediate Filament ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphorylation Site ◽  
Limited Proteolysis ◽  
Complete Removal ◽  
Product Inhibition ◽  
Nucleic Acid Binding ◽  
Intermediate Filament Proteins

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.

POSSIBLE ROLE FOR THE CA2+-DEPENDENT PROTEASE (CALPAIN I) AS AN IRREVERSIBLE ACTIVATOR OF CA2+/CALMODULIN-MEDIATED REACTIONS IN THE HUMAN PLATELET

1987 ◽  
Author(s):  
Robert W Wallace ◽  
E Ann Tallant ◽  
Lynn M Brumley
Keyword(s):  
Myosin Light Chain ◽  
Binding Proteins ◽  
Human Platelet ◽  
Limited Proteolysis ◽  
Physiological Role ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protease Activation ◽  
Stimulation Of

Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.

scholarly journals Structural Stabilization of Human Transthyretin by Centella asiatica (L.) Urban Extract: Implications for TTR Amyloidosis

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 128 ◽  
Author(s):  
Fredrick Nwude Eze ◽  
Ladda Leelawatwattana ◽  
Porntip Prapunpoj
Keyword(s):  
Protein Misfolding ◽  
Quaternary Structure ◽  
Centella Asiatica ◽  
Structural Stabilization ◽  
Flight Mass Spectrometry ◽  
Transmission Electron ◽  
Small Molecule Ligands ◽  
Therapeutic Development ◽  
The Stability

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

Physico-Chemical Studies of Isolated Chromatin Compared with in situ Chromatin after Partial Hepatectomy in the Rat

1979 ◽  
Vol 34 (5-6) ◽  
pp. 442-448 ◽  
Author(s):  
Paul Miller ◽  
Walfried A. Linden ◽  
Claudio Nicolini
Keyword(s):  
Partial Hepatectomy ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Cultured Cells ◽  
Fluorescence Emission ◽  
Chromatin Organization ◽  
Chromatin Conformation ◽  
Functional Changes

Abstract Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity.Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G1 respect to G0 cells.These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G0 -G1 and G1 -S transitions.

scholarly journals Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.

1990 ◽  
Vol 111 (5) ◽  
pp. 2097-2108 ◽  
Author(s):  
K M Weidner ◽  
J Behrens ◽  
J Vandekerckhove ◽  
W Birchmeier
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Human Fibroblasts ◽  
Limited Proteolysis ◽  
Reversed Phase ◽  
Molecular Characteristics ◽  
Collagen Gels ◽  
Scatter Factor ◽  
Human Carcinoma

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We have previously shown that MDCK epithelial cells, which are deprived of intracellular adhesion by the addition of anti-Arc-1/uvomorulin antibodies, become invasive for collagen gels and embryonal heart tissue (Behrens, J., M. M. Mareel, F. M. Van Roy, and W. Birchmeier. 1989. J. Cell Biol. 108: 2435-2447.). Here we examined whether invasiveness is also induced by scatter factor, which is known to dissociate epithelial cells (Stoker, M., E. Gherardi, M. Perryman, and J. Gray. 1987. Nature (Lond.). 327:239-242.). Scatter factor was purified to homogeneity from conditioned medium of human fibroblasts by heparin-Sepharose chromatography, followed by cation exchange chromatography, gel filtration, or preparative SDS gel electrophoresis. We found that scatter factor represents a 92,000 mol wt glycoprotein which, apparently, is converted by limited proteolysis into disulfide-linked 62,000 and 34/32,000 mol wt subunits. Reversed phase HPLC and sequence analysis of tryptic peptides confirmed the suggested molecular structure, and revealed further that scatter factor exhibits sequence similarities to hepatocyte growth factor and to plasminogen. Purified scatter factor in fact induces the invasiveness into collagen matrices of MDCK epithelial cells, and induces or promotes the invasiveness of a number of human carcinoma cell lines. Apparently, the effect on the human cells depends on their respective degree of differentiation, i.e., cell lines with a less pronounced epithelial phenotype were more susceptible to the factor. Scatter factor does not seem to influence synthesis, steady-state level, and phosphorylation of the cell adhesion molecule Arc-1/uvomorulin. Thus, scatter factor represents a clearly defined molecular species which induces, in vitro, the progression of epithelial cells to a more motile, i.e., invasive phenotype.

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