Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β and γ secretases leads to the generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aβ42:Aβ40 ratio and the accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations—M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

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Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations

10.20944/preprints202111.0564.v1 ◽

2021 ◽

Author(s):

Meewhi Kim ◽

Ilya Bezprozvanny

Keyword(s):

Transmembrane Domain ◽

Critical Role ◽

Conformational Flexibility ◽

Proteolytic Processing ◽

Gamma Secretase ◽

App Processing ◽

Late Endosomes ◽

Early Endosomes ◽

Curved Membranes

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in pathogenesis of Azheimer’s disease (AD). Sequential cleavage of APP by β and γ secretases leads to generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) pay a role of catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in increased Aβ42:Aβ40 ratio and accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study we performed molecular modeling of APP complex with γ-secretase and analyzed potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures we proposed that APP can form a complex with γ-secretase in 2 potential conformations – M1 and M2. In conformation M1 transmembrane domain of APP forms a contact with perimembrane domain that follows the transmembrane domain 6 (TM6) in PS1 structure. In conformation M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in PS1 structure. By analyzing effects of PS1-FAD mutations on local protein disorder index, we discovered that these mutations increase conformational flexibility of M2 and reduce conformational flexibility of M1. Based on these results we proposed that M2 conformation, but not M1 conformation, of γ secretase complex with APP leads to amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by a curved membranes, such as membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by a relatively flat memranes such as membranes of late endosomes and plasma membrane. These predictions are consistent with published biochemical analysis of APP processing at different subcellular locations. Our results suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting M2 conformation of γ secretase complex with APP.

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Proteolytic processing of the amyloid-beta protein precursor of Alzheimer's disease

Essays in Biochemistry ◽

10.1042/bse0380037 ◽

2002 ◽

Vol 38 ◽

pp. 37-49 ◽

Cited By ~ 27

Author(s):

Janelle Nunan ◽

David H Small

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amyloid Beta ◽

Alternative Pathway ◽

Protein A ◽

Proteolytic Processing ◽

Gamma Secretase ◽

Amyloid Beta Protein ◽

Protein Precursor ◽

Amyloid Beta Protein Precursor

The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer's disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer's disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer's disease.

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A critical role of the T3SS effector EseJ in intracellular trafficking and replication ofEdwardsiella piscicidain non-phagocytic cells

10.1101/483032 ◽

2018 ◽

Author(s):

Lingzhi Zhang ◽

Jiatiao Jiang ◽

Tianjian Hu ◽

Jin Zhang ◽

Xiaohong Liu ◽

...

Keyword(s):

Bacterial Colonization ◽

Critical Role ◽

Host Cells ◽

Intracellular Replication ◽

Phagocytic Cells ◽

T3ss Effector ◽

Early Endosomes ◽

Underlying Mechanisms ◽

Endosome Maturation

AbstractEdwardsiella piscicida(E. piscicida) is an intracellular pathogen within a broad spectrum of hosts. Essential toE. piscicidavirulence is its ability to survive and replicate inside host cells, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here, we characterized its intracellular lifestyle in non-phagocytic cells and showed that intracellular replication ofE. piscicidain non-phagocytic cells is dependent on its type III secretion system. Following internalization,E. piscicidais contained in vacuoles that transiently mature into early endosomes, but subsequently bypasses the classical endosome pathway and fusion with lysosomes which depends on its T3SS. Following a rapid escape from the degradative pathway,E. piscicidawas found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. We also found that a T3SS effector EseJ is responsible for intracellular replication ofE. piscicidaby preventing endosome/lysosome fusion. Furthermore,in vivoexperiments confirmed that EseJ is necessary for bacterial colonization ofE. piscicidain both mice and zebrafish. Thus, this work elucidates the strategies used byE. piscicidato survive and proliferate within host non-phagocytic cells.Author summaryE. piscicidais a facultative intracellular bacterium associated with septicemia and fatal infections in many animals, including fish and humans. However, little is known about its intracellular life, which is important for successful invasion of the host. The present study is the first comprehensive characterization ofE. piscicida’s intracellular life-style in host cells. Upon internalization,E. piscicidais transiently contained in Rab5-positive vacuoles, but the pathogen prevents further endosome maturation and fusion with lysosomes by utilizing an T3SS effector EseJ. In addition, the bacterium creates an specialized replication niche for rapid growth via an interaction with the ER. Our study provides new insights into the strategies used byE. piscicidato successfully establishes an intracellular lifestyle that contributes to its survival and dissemination during infection.

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Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

10.1101/2020.08.03.235630 ◽

2020 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Hui Wu ◽

Hong Yang ◽

Xue-Hui Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Antiviral Treatment ◽

Critical Role ◽

Virion Assembly ◽

Potential Target ◽

Nuclear Egress ◽

Early Endosomes ◽

Hcmv Replication ◽

Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

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Neuronal aging potentiates beta-amyloid generation via amyloid precursor protein endocytosis

10.1101/616540 ◽

2019 ◽

Author(s):

Tatiana Burrinha ◽

Ricardo Gomes ◽

Ana Paula Terrasso ◽

Cláudia Guimas Almeida

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amyloid Precursor Protein ◽

Precursor Protein ◽

App Processing ◽

Early Endosomes ◽

Primary Mouse ◽

Β Amyloid ◽

Aβ Production

AbstractAging increases the risk of Alzheimer’s disease (AD). During normal aging synapses decline and β-Amyloid (Aβ) accumulates. An Aβ defective clearance with aging is postulated as responsible for Aβ accumulation, although a role for increased Aβ production with aging can also lead to Aβ accumulation. To test this hypothesis, we established a long-term culture of primary mouse neurons that mimics neuronal aging (lysosomal lipofuscin accumulation and synapse decline). Intracellular endogenous Aβ42 accumulated in aged neurites due to increased amyloid-precursor protein (APP) processing. We show that APP processing is up-regulated by a specific age-dependent increase in APP endocytosis. Endocytosed APP accumulated in early endosomes that, in turn were found augmented in aged neurites. APP processing and early endosomes up-regulation was recapitulated in vivo. Finally, we found that inhibition of Aβ production reduced the decline in synapses in aged neurons. We propose that potentiation of APP endocytosis by neuronal aging increases Aβ production, which contributes to aging-dependent decline in synapses.SummaryHow aging increases the risk of Alzheimer’s disease is not clear. We show that normal neuronal aging increases the intracellular production of β-amyloid, due to an upregulation of the amyloid precursor protein endocytosis. Importantly, increased Aβ production contributes to the aging-dependent synapse loss.

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Identification of a putative binding site critical for general anesthetic activation of TRPA1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618144114 ◽

2017 ◽

Vol 114 (14) ◽

pp. 3762-3767 ◽

Cited By ~ 25

Author(s):

Hoai T. Ton ◽

Thieu X. Phan ◽

Ara M. Abramyan ◽

Lei Shi ◽

Gerard P. Ahern

Keyword(s):

Ion Channels ◽

Transient Receptor Potential ◽

Transmembrane Domain ◽

Halogen Bond ◽

Critical Role ◽

Sensory Nerves ◽

Structural Basis ◽

General Anesthetics ◽

General Anesthetic ◽

Potential Binding

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

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Abstract 113: Renal Phenotype of Inwardly Rectifying Potassium Channel Kcnj16 (Kir 5.1) Knockout in the Dahl Salt-Sensitive Rats

Hypertension ◽

10.1161/hyp.66.suppl_1.113 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Oleg Palygin ◽

Vladislav Levchenko ◽

Daria V Ilatovskaya ◽

Jessica L Barnett ◽

Aron M Geurts ◽

...

Keyword(s):

Transmembrane Domain ◽

Basolateral Membrane ◽

Critical Role ◽

Electrolyte Imbalance ◽

Electrophysiological Recording ◽

Resting Membrane Potential ◽

Electrolyte Balance ◽

Renal Tubules ◽

Basolateral Membranes ◽

Hypokalemic Alkalosis

The inward-rectifying channels play an important role in the control of resting membrane potential and tubular homeostasis in the kidney. Kcnj16 (Kir 5.1) form a heteromeric channel with Kcnj10 (Kir 4.1) at the basolateral membranes of aldosterone-sensitive distal nephron (ASDN); mutations in the human KCNJ10 gene result in SeSAME)/EAST syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. To illuminate the importance of Kcnj16 (Kir 5.1) in the context of a disease state in vivo, we generated a Kcnj16 knockout rat model in Dahl salt-sensitive (SS) background by using ZFN technology. ZFN against Kcnj16 caused a 18-bp in-frame deletion that occurred in the second protein transmembrane domain. IHC analysis demonstrated highly specific expression of Kcnj16 on the basolateral membranes of ASDN in the control kidneys of SS rats, which was completely abolished in Kcnj16-/- rats. The electrophysiological recording of K+ channels in the CCD basolateral membrane revealed activity of only homomeric Kcnj10 channels (21 pS channel in Kcnj16-/- rats compared to both 41 and 21 pS channels in SS rats). Thus, these data provide evidence of successful knock out of this protein and consequent degradation of the channel in renal tubules. The Kcnj16-/- knockout in SS rat induces electrolyte imbalance, epileptic seizures and result in changes in development (37% reduction in body and 54% in kidney mass). The mean arterial pressure was significantly lower in Kcnj16-/- compared to SS rats (91.3±1.8 to 104.7±5.5 mmHg) when animals were fed a low salt (0.4%) diet. Knockout of Kcnj16 resulted in hypokalemia (4.25±0.09 vs 2.08±0.12 mmol/L in serum of control vs KO rats), hypermagnesemia (0.49±0.02 vs 0.63±0.01 mmol/L in serum of control vs KO rats), and FSGS. Urea electrolyte balance was also disturbed compared to control animals. Importantly, change of the diet to high salt (4%) caused mortality of KO rats within 1-2 days. These data demonstrate critical role of Kcnj16 channels in renal salt handling and in the development of salt-sensitive hypertension.

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Effects of SEL-12 presenilin on LIN-12 localization and function in Caenorhabditis elegans

Development ◽

10.1242/dev.125.18.3599 ◽

1998 ◽

Vol 125 (18) ◽

pp. 3599-3606 ◽

Cited By ~ 4

Author(s):

D. Levitan ◽

I. Greenwald

Keyword(s):

Plasma Membranes ◽

General Factor ◽

Missense Mutations ◽

Protein Activity ◽

App Processing ◽

Notch Activity ◽

C Elegans ◽

Vulval Precursor Cells ◽

Genes Encoding ◽

And Function

Presenilins have been implicated in the development of Alzheimer's disease and in facilitating LIN-12/Notch activity. Here, we use genetic methods to explore the relationship between C. elegans LIN-12 and SEL-12 presenilin. Reducing sel-12 activity can suppress the effects of elevated lin-12 activity when LIN-12 is activated by missense mutations but not when LIN-12 is activated by removal of the extracellular and transmembrane domains. These results suggest that SEL-12 does not function downstream of activated LIN-12. An active SEL-12::GFP hybrid protein accumulates in the perinuclear region of the vulval precursor cells (VPCs) of living hermaphrodites, consistent with a localization in endoplasmic reticulum/Golgi membranes; when sel-12 activity is reduced, less LIN-12 protein accumulates in the plasma membranes of the VPCs. Together with the genetic interactions between lin-12 and sel-12, these observations suggest a role for SEL-12 in LIN-12 processing or trafficking. However, SEL-12 does not appear to be a general factor that influences membrane protein activity, since reducing sel-12 activity does not suppress or enhance hypomorphic mutations in other genes encoding membrane proteins. We discuss potential parallels for the role of SEL-12/presenilin in facilitating LIN-12/Notch activity and in amyloid precursor protein (APP) processing.

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Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives

Cells ◽

10.3390/cells9061503 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1503 ◽

Cited By ~ 7

Author(s):

Gillian Moore ◽

Stephanie Annett ◽

Lana McClements ◽

Tracy Robson

Keyword(s):

Critical Role ◽

Acquired Resistance ◽

Tumour Microenvironment ◽

Phase Iii ◽

Cell Renewal ◽

Gamma Secretase ◽

Cancer Hallmarks ◽

Activating Mutations ◽

Stem Cell Renewal ◽

Exciting Time

Evolutionarily conserved Notch plays a critical role in embryonic development and cellular self-renewal. It has both tumour suppressor and oncogenic activity, the latter of which is widely described. Notch-activating mutations are associated with haematological malignancies and several solid tumours including breast, lung and adenoid cystic carcinoma. Moreover, upregulation of Notch receptors and ligands and aberrant Notch signalling is frequently observed in cancer. It is involved in cancer hallmarks including proliferation, survival, migration, angiogenesis, cancer stem cell renewal, metastasis and drug resistance. It is a key component of cell-to-cell interactions between cancer cells and cells of the tumour microenvironment, such as endothelial cells, immune cells and fibroblasts. Notch displays diverse crosstalk with many other oncogenic signalling pathways, and may drive acquired resistance to targeted therapies as well as resistance to standard chemo/radiation therapy. The past 10 years have seen the emergence of different classes of drugs therapeutically targeting Notch including receptor/ligand antibodies, gamma secretase inhibitors (GSI) and most recently, the development of Notch transcription complex inhibitors. It is an exciting time for Notch research with over 70 cancer clinical trials registered and the first-ever Phase III trial of a Notch GSI, nirogacestat, currently at the recruitment stage.

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Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/6927380 ◽

2019 ◽

Vol 2019 ◽

pp. 1-21 ◽

Cited By ~ 2

Author(s):

Matylda B. Mielcarska ◽

Magdalena Bossowska-Nowicka ◽

Karolina P. Gregorczyk-Zboroch ◽

Zbigniew Wyżewski ◽

Lidia Szulc-Dąbrowska ◽

...

Keyword(s):

Immune Responses ◽

Signaling Pathway ◽

Critical Role ◽

Antiviral Response ◽

Proteolytic Processing ◽

Viral Dsrna ◽

Kinase Substrate ◽

Tlr3 Signaling ◽

The Brain

Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-β, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-β by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer’s disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.

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