scholarly journals The evolved divergence of γ-secretase-susceptibility of homologous proteins Ngfrb and Nradd in zebrafish

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tanya Jayne ◽  
Morgan Newman ◽  
Lachlan Baer ◽  
Michael Lardelli
Keyword(s):  
Transmembrane Domain ◽  
Chimeric Protein ◽  
Differential Susceptibility ◽  
Duplication Event ◽  
Zebrafish Embryos ◽  
Structural Constraints ◽  
Homologous Proteins ◽  
Western Immunoblotting ◽  
Secretase Activity ◽  
The Stability

Abstract Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.

P1-169: Transmembrane domain of CRB2 is indispensable for inhibition of γ-secretase activity

2009 ◽  
Vol 5 (4S_Part_8) ◽  
pp. P226-P227
Author(s):  
Masaki Nishimura ◽  
Yachiyo Mitsuishi ◽  
Hiroshi Hasegawa ◽  
Akinori Matsuo ◽  
Shinji Tagami ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Secretase Activity

scholarly journals STUDY OF A STRUCTURE SUSPENDED FROM A SYSTEM OF STEEL COLUMNS AND TRUSSES (SYSTEM AND STABILITY)

2020 ◽  
Vol 9 (9) ◽  
pp. 43-54
Keyword(s):  
Reinforced Concrete ◽  
Lateral Force ◽  
Concrete Slabs ◽  
Structural Constraints ◽  
Steel Columns ◽  
Force Method ◽  
Reinforced Concrete Slabs ◽  
The Stability

Throughout history, men always wanted to build structures that are each more impressive than the next, while rising higher in the air. In this process, men were not satisfied with making sure that these structures were beautiful, impressive and majestic, but that they could also be very useful, that they fulfilled a function, and that they were able to resist the various structural constraints that will be imposed on it, or that could be imposed on it. With this in mind, we thought of creating a structure that could both inspire this side of wonder and structural beauty, while being useful and resistant to the loads imposed on it. In this work, we are going to talk about a building suspended to its foundation, in the sense that the building does not rest directly on the ground, but is suspended nearly eight meters from the ground by each of the three columns which support the said building by a system of trusses. The structure is made of steel with reinforced concrete slabs, which gives it a significant advantage in terms of weight. Another advantage is that it reacts quite well to earthquakes, showing only very small deflections using the equivalent lateral force method. In this work we will focus on the stability of the members of the system that carries the building and the stability of the building in general.

scholarly journals Amino- and Carboxy-Terminal PEST Domains Mediate Gastrin Stabilization of Rat l-Histidine Decarboxylase Isoforms

2000 ◽  
Vol 20 (13) ◽  
pp. 4932-4947 ◽  
Author(s):  
John V. Fleming ◽  
Timothy C. Wang
Keyword(s):  
Histidine Decarboxylase ◽  
Chimeric Protein ◽  
Proteasome Inhibition ◽  
Peptide Hormone ◽  
Mrna Levels ◽  
Intracellular Targeting ◽  
Enzymatic Function ◽  
Steady State Levels ◽  
The Stability ◽  
Carboxy Terminal

ABSTRACT Control of enzymatic function by peptide hormones can occur at a number of different levels and can involve diverse pathways that regulate cleavage, intracellular trafficking, and protein degradation. Gastrin is a peptide hormone that binds to the cholecystokinin B-gastrin receptor and regulates the activity ofl-histidine decarboxylase (HDC), the enzyme that produces histamine. Here we show that gastrin can increase the steady-state levels of at least six HDC isoforms without affecting HDC mRNA levels. Pulse-chase experiments indicated that HDC isoforms are rapidly degraded and that gastrin-dependent increases are due to enhanced isoform stability. Deletion analysis identified two PEST domains (PEST1 and PEST2) and an intracellular targeting domain (ER2) which regulate HDC protein expression levels. Experiments with PEST domain fusion proteins demonstrated that PEST1 and PEST2 are strong and portable degradation-promoting elements which are positively regulated by both gastrin stimulation and proteasome inhibition. A chimeric protein containing the PEST domain of ornithine decarboxylase was similarly affected, indicating that gastrin can regulate the stability of other PEST domain-containing proteins and does so independently of antizyme/antizyme inhibitor regulation. At the same time, endoplasmic reticulum localization of a fluorescent chimera containing the ER2 domain of HDC was unaltered by gastrin stimulation. We conclude that gastrin stabilization of HDC isoforms is dependent upon two transferable and sequentially unrelated PEST domains that regulate degradation. These experiments revealed a novel regulatory mechanism by which a peptide hormone such as gastrin can disrupt the degradation function of multiple PEST-domain-containing proteins.

Functional analysis and molecular model of the human urate transporter/channel, hUAT

2002 ◽  
Vol 283 (1) ◽  
pp. F150-F163 ◽  
Author(s):  
Edgar Leal-Pinto ◽  
B. Eleazar Cohen ◽  
Michael S. Lipkowitz ◽  
Ruth G. Abramson
Keyword(s):  
Functional Analysis ◽  
Lipid Bilayers ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Molecular Model ◽  
Open Probability ◽  
Homologous Proteins ◽  
Urate Transporter ◽  
Binding Domains

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two β-galactoside binding domains that bind lactose. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.

scholarly journals In SilicoIdentification of Functional Protein Interfaces

2003 ◽  
Vol 4 (4) ◽  
pp. 420-423 ◽  
Author(s):  
Rachel E. Bell ◽  
Nir Ben-Tal
Keyword(s):  
Phylogenetic Trees ◽  
Large Scale ◽  
Hypothetical Protein ◽  
Evolutionary Information ◽  
Structural Constraints ◽  
Functional Protein ◽  
Homologous Proteins ◽  
Geometrical Properties ◽  
Protein Interfaces ◽  
Branch Lengths

Proteins perform many of their biological roles through protein–protein, protein–DNA or protein–ligand interfaces. The identification of the amino acids comprising these interfaces often enhances our understanding of the biological function of the proteins. Many methods for the detection of functional interfaces have been developed, and large-scale analyses have provided assessments of their accuracy. Among them are those that consider the size of the protein interface, its amino acid composition and its physicochemical and geometrical properties. Other methods to this effect use statistical potential functions of pairwise interactions, and evolutionary information. The rationale of the evolutionary approach is that functional and structural constraints impose selective pressure; hence, biologically important interfaces often evolve at a slower pace than do other external regions of the protein. Recently, an algorithm, Rate4Site, and a web-server, ConSurf (http://consurf.tau.ac.il/), for the identification of functional interfaces based on the evolutionary relations among homologous proteins as reflected in phylogenetic trees, were developed in our laboratory. The explicit use of the tree topology and branch lengths makes the method remarkably accurate and sensitive. Here we demonstrate its potency in the identification of the functional interfaces of a hypothetical protein, the structure of which was determined as part of the international structural genomics effort. Finally, we propose to combine complementary procedures, in order to enhance the overall performance of methods for the identification of functional interfaces in proteins.

scholarly journals Membrane-anchored metalloprotease MDC9 has an α-secretase activity responsible for processing the amyloid precursor protein

1999 ◽  
Vol 343 (2) ◽  
pp. 371-375 ◽  
Author(s):  
Hisashi KOIKE ◽  
Shigeo TOMIOKA ◽  
Hiroyuki SORIMACHI ◽  
Takaomi C. SAIDO ◽  
Kei MARUYAMA ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Phorbol Ester ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Sequence Similarity ◽  
Precursor Protein ◽  
Cos Cells ◽  
Secretase Activity ◽  
Epidermal Growth ◽  
Metalloprotease Inhibitor

MDC9, also known as meltrin γ, is a membrane-anchored metalloprotease. MDC9 contains several distinct protein domains: a signal sequence followed by a prodomain and a domain showing sequence similarity to snake venom metalloproteases, a disintegrin-like domain, a cysteine-rich region, an epidermal-growth-factor-like repeat, a transmembrane domain and a cytoplasmic domain. Here we demonstrate that MDC9 expressed in COS cells is cleaved between the prodomain and the metalloprotease domain. Further, when MDC9 was co-expressed in COS cells with amyloid precursor protein (APP695) and treated with phorbol ester, APP695 was digested exclusively at the α-secretory site in MDC9-expressing cells. When an artificial α-secretory site mutant was also co-expressed with MDC9 and treated with phorbol ester, APP secreted by α-secretase was not increased in conditional medium. Inhibition of MDC9 by a hydroxamate-based metalloprotease inhibitor, SI-27, enhanced β-secretase cleavage. These results suggest that MDC9 has an α-secretase-like activity and is activated by phorbol ester.

scholarly journals PC8 [corrected], a new member of the convertase family

1996 ◽  
Vol 314 (3) ◽  
pp. 727-731 ◽  
Author(s):  
Angela BRUZZANITI ◽  
Katrina GOODGE ◽  
Philippe JAY ◽  
Sylvie A. TAVIAUX ◽  
Mark H. C. Lam ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Duplication Event ◽  
Amino Acid Identity ◽  
Chromosome 15 ◽  
Rat Tissues ◽  
Amino Acid Residues ◽  
Degenerate Primers ◽  
Chromosome 11Q23 ◽  
Ancient Gene Duplication

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23–11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

Processive proteolysis by γ-secretase and the mechanism of Alzheimer’s disease

2012 ◽  
Vol 393 (9) ◽  
pp. 899-905 ◽  
Author(s):  
Michael S. Wolfe
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Transmembrane Domain ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Amyloid Β Protein ◽  
Loss Of Function ◽  
Gain Of Function ◽  
C Terminus ◽  
Secretase Activity

Abstract γ-Secretase is a membrane-embedded protease complex with presenilin as the catalytic component. Cleavage within the transmembrane domain of the amyloid β-protein precursor (APP) by γ-secretase produces the C-terminus of the amyloid β-peptide (Aβ), a proteolytic product prone to aggregation and strongly linked to Alzheimer’s disease (AD). Presenilin mutations are associated with early-onset AD, but their pathogenic mechanisms are unclear. One hypothesis is that these mutations cause AD through a toxic gain of function, changing γ-secretase activity to increase the proportion of 42-residue Aβ over the more soluble 40-residue form. A competing hypothesis is that the mutations cause AD through a loss of function, by reducing γ-secretase activity. However, γ-secretase apparently has two types of activities, an endoproteolytic function that first cuts APP to generate a 48/49-residue form of Aβ, and a carboxypeptidase activity that processively trims these longer Aβ intermediates approximately every three residues to form shorter, secreted forms. Recent studies suggest a resolution of the gain-of-function vs. loss-of-function debate: presenilin mutations may increase the proportion of longer, more aggregation-prone Aβ by specifically decreasing the trimming activity of γ-secretase. That is, the reduction of this particular proteolytic function of presenilin, not its endoproteolytic activity, may lead to the neurotoxic gain of function.

scholarly journals Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Chloé Habermacher ◽  
Adeline Martz ◽  
Nicolas Calimet ◽  
Damien Lemoine ◽  
Laurie Peverini ◽  
...  
Keyword(s):  
Bound States ◽  
Transmembrane Domain ◽  
P2x Receptors ◽  
Structural Constraints ◽  
Molecular Tweezers ◽  
Gating Mechanism ◽  
Outer Pore ◽  
Pore Opening ◽  
High Resolution Models ◽  
Hinge Bending

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

scholarly journals Assessment of Structural Units Deletions in the Archaeal Oligosaccharyltransferase AglB

2020 ◽  
Author(s):  
Conrado Pedebos ◽  
Hugo Verli
Keyword(s):  
Structural Integrity ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Wild Type Enzyme ◽  
Structural Units ◽  
Terminal Domain ◽  
Glycan Chain ◽  
The Stability ◽  
Dynamics Simulations ◽  
Binding Interfaces

AbstractOligosaccharyltransferases (OSTs) are enzymes that catalyze the transfer of a glycan chain to an acceptor protein. Their structure is composed by a transmembrane domain and a periplasmic / C-terminal domain, which can be divided into structural units. The Archaeoglobus fulgidus OST, AfAglB, has unique structural units with unknown functions. Here, we evaluate the stability role proposed for AfAglB units by employing molecular modelling and molecular dynamics simulations, to examine the effect of single and double deletions in the enzyme structure. Our results show a strong effect on the dynamics of the C-terminal domain for the mutated systems with increased fluctuations near the deleted areas. Conformational profile and stability are deeply affected, mainly in the double unit deletion, modifying the enzyme behavior and binding interfaces. Coordination at the catalytic site was not disrupted, indicating that the mutated enzymes could retain activity at some level. Hotspots of variation were identified and rationalized with previous data. Our data shows that structural units may provide stabilization interactions, contributing for integrity of the wild-type enzyme at high temperatures. By correlating our findings to structural units mutagenesis experimental data available, it was observed that structural units deletion can interfere with OSTs stability and dynamics but it is not directly related to catalysis. Instead, they may influence the OST structural integrity, and, potentially, thermostability. This work offers a basis for future experiments involving OSTs structural and functional characterization, as well as for protein engineering.

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