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 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.

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 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.

scholarly journals Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

2004 ◽  
Vol 166 (7) ◽  
pp. 1041-1054 ◽  
Author(s):  
Cary Esselens ◽  
Viola Oorschot ◽  
Veerle Baert ◽  
Tim Raemaekers ◽  
Kurt Spittaels ◽  
...  
Keyword(s):  
Half Life ◽  
Cathepsin D ◽  
Hippocampal Neurons ◽  
Transmembrane Domain ◽  
Presenilin 1 ◽  
Wild Type ◽  
Degradative Pathway ◽  
Autophagic Vacuoles ◽  
Secretase Activity ◽  
Lysosomal Proteins

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579–589). Here, we demonstrate that TLN is not a substrate for γ-secretase cleavage, but displays a prolonged half-life in PS1−/− hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from γ-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a γ-secretase–independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.

scholarly journals Rer1p competes with APH-1 for binding to nicastrin and regulates γ-secretase complex assembly in the early secretory pathway

2007 ◽  
Vol 176 (5) ◽  
pp. 629-640 ◽  
Author(s):  
Dragana Spasic ◽  
Tim Raemaekers ◽  
Katleen Dillen ◽  
Ilse Declerck ◽  
Veerle Baert ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Limiting Factor ◽  
Type I ◽  
Complex Assembly ◽  
Early Secretory Pathway ◽  
Regulated Intramembrane Proteolysis ◽  
Secretase Activity ◽  
Therapeutic Value ◽  
Competitive Nature

The γ-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this “proteasome of the membrane” are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of γ-secretase subcomplexes and, concomitantly, total cellular γ-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular γ-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value.

scholarly journals Effect of Metal Chelators onγ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Michael Ho ◽  
David E. Hoke ◽  
Yee Jia Chua ◽  
Qiao-Xin Li ◽  
Janetta G. Culvenor ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Size Exclusion ◽  
Magnesium Ions ◽  
Gamma Secretase ◽  
Metal Chelators ◽  
Human Neuroblastoma ◽  
Secretase Activity ◽  
Metalloprotease Inhibitors ◽  
Calcium And Magnesium ◽  
Calcium And Magnesium Ions

Gamma-secretase is involved in the production of Aβamyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβand the APP intracellular domain (AICD). Metal ions play an important role in Aβaggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators onγ-secretase has not yet been investigated. The authors used anin vitro γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenousγ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect onγ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreasedγ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg2+stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+and Mg2+stabilizeγ-secretase and enhance its activity.

scholarly journals The Extreme C Terminus of Presenilin 1 Is Essential for γ-Secretase Complex Assembly and Activity

2004 ◽  
Vol 279 (44) ◽  
pp. 45564-45572 ◽  
Author(s):  
Anna Bergman ◽  
Hanna Laudon ◽  
Bengt Winblad ◽  
Johan Lundkvist ◽  
Jan Näslund
Keyword(s):  
Transmembrane Domain ◽  
Amyloid Β ◽  
Presenilin 1 ◽  
Amyloid Β Peptide ◽  
Terminal Part ◽  
Complex Assembly ◽  
Terminal Fragment ◽  
C Terminus ◽  
Secretase Activity ◽  
Endoproteolytic Cleavage

The γ-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid β-peptide and the cytoplasmic APP intracellular domain. The active γ-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for γ-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, γ-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease γ-secretase activity, PS1 endoproteolysis, and γ-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.

scholarly journals Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

2021 ◽  
Author(s):  
Sanjay Bhattarai ◽  
Sujan Devkota ◽  
Michael S Wolfe
Keyword(s):  
Transition State ◽  
Active Site ◽  
Transmembrane Domain ◽  
Competitive Inhibition ◽  
Solid Phase ◽  
Substrate Recognition ◽  
Solid Phase Peptide Synthesis ◽  
Rational Drug Design ◽  
Binding Assays ◽  
Secretase Activity

γ-Secretase, a membrane-embedded aspartyl protease complex with presenilin as the catalytic component, cleaves within the transmembrane domain (TMD) of its many substrates, which include the amyloid precursor protein (APP) of Alzheimer's disease (AD). APP TMD is processively cut by γ-secretase through endoproteolysis followed by tricarboxypeptidase "trimming", the latter function being deficient with mutations causing hereditary AD. Toward better understanding of substrate recognition and hydrolytic reactions catalyzed by this enzyme within its hydrophobic milieu, we recently developed a prototype substrate TMD mimetic as a chemical tool for structural analysis (Bhattarai S et al. J. Am. Chem. Soc., 2020, 142(7): 3351-3355). This TMD mimetic--composed of a helical peptide inhibitor (HPI) connected through a linker to a transition-state analog inhibitor (TSA)--simultaneously engages the substrate docking exosite and the active site and is pre-organized to trap the protease transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate. New HPI-TSA conjugates were synthesized using an earlier established divergent strategy, which involved the construction of a tripeptidomimetic building block followed by solid-phase peptide synthesis (SPPS). For each class of structural probe, SAR analysis led to discovery of highly potent prototypes with stoichiometric or low-nanomolar inhibition. These TMD mimetics exhibited non-competitive inhibition of γ-secretase activity and occupy both docking and active site as demonstrated by enzyme cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

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