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Hidradenitis suppurativa (HS) is a chronic skin disease characterized by painful, recurrent abscesses, nodules, and scarring, primarily in skin folds. The exact causes of HS are multifactorial, involving genetic, hormonal, and environmental factors. It is associated with systemic diseases such as metabolic syndrome and inflammatory bowel disease. Genetic studies have identified mutations in the γ-secretase complex that affect Notch signaling pathways critical for skin cell regulation. Despite its high heritability, most reported HS cases do not follow a simple genetic pattern. In this article, we performed whole-exome sequencing (WES) on a cohort of 100 individuals with HS, and we provide a comprehensive review of the variants known to be described or associated with HS. 91 variants were associated with the γ-secretase complex, and 78 variants were associated with other genes involved in the Notch pathway, keratinization, or immune response. Through this new genetic analysis, we have added ten new variants to the existing catalogs. All variants are available in a.vcf file and are provided as a resource for future studies. [ABSTRACT FROM AUTHOR]

Gamma secretase (GS) is an intramembranous enzyme that acts on the amyloid precursor protein and Notch inside lipid membranes. The enzyme is responsible for amyloid-β propagation, one of the well-known causes of Alzheimer's disease. However, the effects of lipids on GS activity and structural dynamics are unknown. Therefore, in this study, we performed coarse-grained molecular dynamics simulations to probe the effects of five individual lipids on GS. These lipids included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-phosphatidylcholine (DOPC), 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). These lipids are structurally characterized by different heads (i.e., NH3 [PE] for POPE vs. NC3 [PC] for POPC), number of double bonds (one for POPC vs. two for DOPC), and alkyl tail chain lengths (16:1/18:1 for DOPC vs. 14:0/14:0 for DMPC vs. 12:0/12:0 for DLPC). This indicates distinct microenvironments and adjustable structural elements for catalytic function when GS is embedded. Our results revealed that the presence of more unsaturated bonds in DOPC than in POPC resulted in greater GS stability. Moreover, lipids with short alkyl tail chains or with PC heads instead of PE heads had improved mobility of the sixth transmembrane helix of GS, which is responsible for the considerable active site flexibility and presenilin 1 subunit plasticity. The length of the DMPC alkyl tail chain was between that of DOPC and DLPC because the up-down and cross-correlation motions of GS in DMPC was the lowest among the three lipids, and GS mobility in DMPC was the lowest among all five lipids. This may be because the alkyl tail chain length (i.e., 3. 8 nm thickness of the DMPC bilayer) was suitable for GS embedding, thereby restraining more GS motions than that of the long (DOPC) or short lipids (DLPC). Collectively, these results indicated that GS activity can be modulated through changes in conformational fluctuations, structural perturbations, molecular motion, and cross-correlation motion when embedded in different lipids. Exploration of such fundamental information can reveal the possible mechanisms by which GS is affected by individual lipid species.

Gamma secretase (GS) is an intramembranous enzyme that acts on the amyloid precursor protein and Notch inside lipid membranes. The enzyme is responsible for amyloid-β propagation, one of the well-known causes of Alzheimer's disease. However, the effects of lipids on GS activity and structural dynamics are unknown. Therefore, in this study, we performed coarse-grained molecular dynamics simulations to probe the effects of five individual lipids on GS. These lipids included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-phosphatidylcholine (DOPC), 2-dimyristoyl-snglycero-3-phosphocholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). These lipids are structurally characterized by different heads (i.e., NH3 [PE] for POPE vs. NC3 [PC] for POPC), number of double bonds (one for POPC vs. two for DOPC), and alkyl tail chain lengths (16:1/18:1 for DOPC vs. 14:0/14:0 for DMPC vs. 12:0/12:0 for DLPC). This indicates distinct microenvironments and adjustable structural elements for catalytic function when GS is embedded. Our results revealed that the presence of more unsaturated bonds in DOPC than in POPC resulted in greater GS stability. Moreover, lipids with short alkyl tail chains or with PC heads instead of PE heads had improved mobility of the sixth transmembrane helix of GS, which is responsible for the considerable active site flexibility and presenilin 1 subunit plasticity. The length of the DMPC alkyl tail chain was between that of DOPC and DLPC because the up-down and cross-correlation motions of GS in DMPC was the lowest among the three lipids, and GS mobility in DMPC was the lowest among all five lipids. This may be because the alkyl tail chain length (i.e., 3. 8 nm thickness of the DMPC bilayer) was suitable for GS embedding, thereby restraining more GS motions than that of the long (DOPC) or short lipids (DLPC). Collectively, these results indicated that GS activity can be modulated through changes in conformational fluctuations, structural perturbations, molecular motion, and cross-correlation motion when embedded in different lipids. Exploration of such fundamental information can reveal the possible mechanisms by which GS is affected by individual lipid species. [ABSTRACT FROM AUTHOR]

Background and purpose: Although some proposed mechanisms responsible for tamoxifen resistance have already been present, further study is needed to determine the mechanisms underlying tamoxifen resistance more clearly. The critical role of Notch signaling has been described in promoting resistance in therapeutics, but there is little information about its role in tamoxifen resistance progression. Experimental approach: In the present study, the expression of Notch pathway genes, including Notch4, nicastrin and the Notch downstream target Hes1 was evaluated using quantitative RT-PCR in 36 tamoxifen-resistant (TAM-R) and 36 tamoxifen-sensitive (TAM-S) patients. Expression data were correlated with the clinical outcome and survival of patients. Findings/Results: mRNA levels of Notch4 (fold change = 2.7), nicastrin (fold change = 6.71), and Hes1 (fold change= 7.07) were significantly higher in TAM-R breast carcinoma patients compared to sensitive cases. We confirmed all these genes were co-expressed. Hence, it seems that Notch signaling is involved in tamoxifen resistance in our TAM-R patients. Obtained results showed that Hes1, nicastrin, and Notch4 mRNA upregulation was correlated with the N stage. The extracapsular nodal extension was associated with nicastrin and Notch4 overexpression. Moreover, nicastrin overexpression was correlated with perineural invasion. Hes1 upregulation was also associated with nipple involvement. Finally, the Cox regression proportional hazard test revealed that overexpression of nicastrin was an independent worse survival factor. Conclusion and implications: Presumably, upregulation of the Notch pathway may be involved in tamoxifen resistance in breast cancer patients.

Background: Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β peptide (Aβ) in the brain. Aβ is produced by sequential β- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting β- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aβ production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aβ generation without inhibiting any enzyme. Methods: An APP mutant mimicking secreted APPγ was overexpressed in cells to test β-cleavage and Aβ production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms. Results: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aβ. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aβ, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase. Conclusions: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aβ. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy.

Summary: Hidradenitis suppurativa (HS) is a chronic, debilitating skin disease for which few treatment options are available. While most HS is sporadic, some rare kindred show a high-penetrance, autosomal-dominant inheritance. We wanted to identify rare variants that could contribute to HS risk in sporadic cases using candidate gene sequencing. We ultimately identified 21 genes for our capture panel. We included genes of the γ-secretase complex (n = 6) because rare variants in these genes sometimes cause familial HS. We added Notch receptor and ligand genes (n = 13) because γ-secretase is critical for processing Notch receptor signaling. Clinically, some people with PAPA (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, a rare inflammatory disease, have concurrent HS. Rare variants in PSTPIP1 are known to cause PAPA syndrome, so we included PSTPIP1 and PSTPIP2 in the capture panel. We screened 117 individuals with HS for rare variations and calculated the expected burden using Genome Aggregation Database (gnomAD) allele frequencies. We discovered two pathogenic loss-of-function variants in NCSTN. This class of NCSTN variant can cause familial HS. There was no increased burden of rare variations in any γ-secretase complex gene. We did find that individuals with HS had a significantly increased number of rare missense variants in the SH3 domain of PSTPIP1. This finding, therefore, implicates PSTPIP1 variation in sporadic HS and further supports dysregulated immunity in HS. Our data also suggests that population-scale HS genetic research will yield valuable insights into disease pathology.

Background: Nicastrin is a confirmed breast cancer target, but the lack of knowledge about its binding sites and the structural basis of interactions with known small molecules makes the development of small molecules against it challenging. Methods: Molecular docking and molecular dynamics simulations were used in this work to identify binding sites in nicastrin, a gamma-secretase component that has been implicated in breast cancer and a potential drug target in cancer chemotherapy. Results: Docking calculations identified three binding sites, however binding site analysis using druggability assessment identified a region that encompasses the DYIGS motif, the DYIGS site as the most favorable binding site. This site was validated by a 50 ns molecular dynamics simulation with a known inhibitor CID44433923 and free energy of binding was found to be -11.4 kcal/mol and mainly driven by hydrophobic interactions. Per residue decomposition analysis showed that Gln139, Val138 and Arg105 had a relatively high contribution towards the free energy of binding. These results suggest that these residues might be critical in nicastrin inhibition. Binding mode analysis by docking previously reported nicastrin inhibitors identified residues Gln139, Val138 and Asp143 as key in the interactions. Conclusions: This work affords an insight into the binding mechanism of small molecules and might direct drug design efforts towards nicastrin.

Hidradenitis suppurativa (HS) is a disease of the pilosebaceous unit characterized by recurrent nodules, abscesses and draining tunnels with a predilection to intertriginous skin. The pathophysiology of HS is complex. However, it is known that inflammation and hyperkeratinization at the hair follicle play crucial roles in disease manifestation. Genetic and environmental factors are considered the main drivers of these two pathophysiological processes. Despite a considerable proportion of patients having a positive family history of disease, only a minority of patients suffering from HS have been found to harbor monogenic variants which segregate to affected kindreds. Most of these variants are in the secretase complex (GSC) protein-coding genes. In this manuscript, we set out to characterize the burden of missense pathogenic variants in healthy reference population using large scale genomic dataset thereby providing a standard for comparing genomic variation in GSC protein-coding genes in the HS patient cohort. [ABSTRACT FROM AUTHOR]

Abstract Alterations in the canonical processing of Amyloid Precursor Protein generate proteoforms that contribute to the onset of Alzheimer’s Disease. Modified composition of γ-secretase or mutations in its subunits has been directly linked to altered generation of Amyloid beta. Despite biochemical evidence about the role of γ-secretase in the generation of APP, the molecular origin of how spatial heterogeneity in the generation of proteoforms arises is not well understood. Here, we evaluated the localization of Nicastrin, a γ-secretase subunit, at nanometer sized functional zones of the synapse. With the help of super resolution microscopy, we confirm that Nicastrin is organized into nanodomains of high molecular density within an excitatory synapse. A similar nanoorganization was also observed for APP and the catalytic subunit of γ-secretase, Presenilin 1, that were discretely associated with Nicastrin nanodomains. Though Nicastrin is a functional subunit of γ-secretase, the Nicastrin and Presenilin1 nanodomains were either colocalized or localized independent of each other. The Nicastrin and Presenilin domains highlight a potential independent regulation of these molecules different from their canonical secretase function. The collisions between secretases and substrate molecules decide the probability and rate of product formation for transmembrane proteolysis. Our observations of secretase nanodomains indicate a spatial difference in the confinement of substrate and secretases, affecting the local probability of product formation by increasing their molecular availability, resulting in differential generation of proteoforms even within single synapses.

Hidradenitis suppurativa (HS) is a disease of the pilosebaceous unit characterized by recurrent nodules, abscesses and draining tunnels with a predilection to intertriginous skin. The pathophysiology of HS is complex. However, it is known that inflammation and hyperkeratinization at the hair follicle play crucial roles in disease manifestation. Genetic and environmental factors are considered the main drivers of these two pathophysiological processes. Despite a considerable proportion of patients having a positive family history of disease, only a minority of patients suffering from HS have been found to harbor monogenic variants which segregate to affected kindreds. Most of these variants are in the ɣ secretase complex (GSC) protein-coding genes. In this manuscript, we set out to characterize the burden of missense pathogenic variants in healthy reference population using large scale genomic dataset thereby providing a standard for comparing genomic variation in GSC protein-coding genes in the HS patient cohort.

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica , however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites. [Display omitted] • This is the first report of the γ-secretase complex in protozoan parasites. • Presenilin and nicastrin are present in the Entamoeba genome. • Presenilin and nicastrin form a complex on the plasma membrane. • The activity of amoebic presenilin was inhibited by a γ-secretase inhibitor. [ABSTRACT FROM AUTHOR]

• Injection of human NCSTN mutations encoding null alleles leads to zebrafish hypopigmentation. • Alterations in melanophore number, size, migration, and distribution are observed. • Phenotype can be rescued by co-injection of zebrafish ncstn and human NCSTN mRNA. • Hypopigmentation is also reversed by co-injection of human NCSTN missense mutation p.P211R. • The zebrafish ncstn gene and the human NCSTN gene may have acquired evolutionarily distinct roles. Mutations in the genes that encode the human γ-secretase subunits Presenilin-1, Presenilin Enhancer Protein 2, and Nicastrin (NCSTN) are associated with familial hidradenitis suppurativa (HS); and, regarding Presenilin Enhancer Protein 2, also with comorbidity for the hereditary pigmentation disorder Dowling-Degos disease. Here, the consequences of targeted inactivation of ncstn, the zebrafish homologue of human NCSTN, were studied. After morpholino (MO)-mediated ncstn -knockdown, the possibilities of phenotype rescue through co-injection of ncstn -MO with wildtype zebrafish ncstn or human NCSTN mRNA were investigated. Further, the effects of the co-injection of a human missense, nonsense, splice-site, and frameshift mutation were studied. MO-mediated ncstn -knockdown resulted in a significant reduction in melanophore morphology, size and number; and alterations in their patterns of migration and distribution. This phenotype was rescued by co-injection of zebrafish ncstn RNA, human NCSTN RNA, or a construct encoding the human NCSTN missense mutation p.P211R. Human NCSTN mutations encoding null alleles confer loss-of-function regarding pigmentation homeostasis in zebrafisch. In contrast, the human missense mutation p.P211R was less harmful, asserting sufficient residual ncstn activity to maintain pigmentation in zebrafish. Since fish lack the anatomical structures affected by HS, our data suggest that the zebrafish ncstn gene and the human NCSTN gene have probably acquired different functions during evolution. In fish, one major role of ncstn is the maintenance of pigmentation homeostasis. In contrast, one of the roles of NCSTN in humans is the prevention of inflammatory processes in the adnexal structures of the skin, as seen in familial HS. [ABSTRACT FROM AUTHOR]

Abstract The activity of γ-secretase is critical to the pathogenesis of Alzheimer’s disease (AD). How its activity is regulated is intriguing and highly important for any AD therapy that focuses on reduction of toxic amyloid peptides and amyloid deposition in patients. Recently, interferon-induced transmembrane protein 3 (IFITM3) has been identified as a novel regulator of γ-secretase through a specific interaction. This commentary highlights this exciting study and provides an updated link of γ-secretase activity to innate immunity through IFITM3.

Abstract Background Hepatocellular carcinoma is the third top cause of cancer-related mortalities worldwide. The prognosis of HCC patients remains poor due to rapid progression and high incidence of tumor recurrence. Nicastrin (NCSTN), a core subunit of γ-Secretase, has been reported to play a vital role in tumor progression. However, no study till now has revealed its role in HCC. Methods The expression of NCSTN was evaluated by immunohistochemical staining, Western blot, and quantitative real-time PCR. Cell counting kit-8, colony formation and cell cycle assays were used for evaluating cell growth in vitro. Transwell and wound-healing assays were used for evaluating cell migration and invasion capacity. Immunofluorescence, subcellular protein fractionation and co-immunoprecipitation were used for location analysis of β-catenin. The in vivo functions of NCSTN were illustrated by xenograft tumor models. Results NCSTN was dramatically overexpressed in HCC compared to normal liver tissues. Elevated NCSTN expression level was significantly correlated to worse overall and recurrence-free survival of HCC patients. Enhanced NCSTN expression promoted HCC cell growth, migration and invasion in vitro and in vivo. Mechanistic investigations showed that NCSTN induced epithelial-mesenchymal transition (EMT) process via upregulation of Zeb1. Subsequently, we revealed that NCSTN facilitated nuclear translocation of β-catenin, a positive transcriptional regulator of Zeb1. Using Notch and AKT inhibitors, we revealed that NCSTN promoted β-catenin activation through Notch1 and AKT signaling pathway. NCSTN increased AKT and GSK-3β phosphorylation by cleavage of Notch1, which decreased GSK-3β/β-catenin complex. The inactivation of GSK-3β inhibited the β-catenin degradation and promoted nuclear translocation of β-catenin to initiate transcription of Zeb1, resulting in malignant phenotype. Conclusions Our results demonstrated that NCSTN promoted HCC cell growth and metastasis via β-catenin-mediated upregulation of Zeb1 in a Notch1/AKT dependent manner, suggesting that NCSTN might serve as a potential prognostic marker and therapeutic target for HCC.

Alterations in the canonical processing of Amyloid Precursor Protein generate proteoforms that contribute to the onset of Alzheimer's Disease. Modified composition of γ-secretase or mutations in its subunits has been directly linked to altered generation of Amyloid beta. Despite biochemical evidence about the role of γ-secretase in the generation of APP, the molecular origin of how spatial heterogeneity in the generation of proteoforms arises is not well understood. Here, we evaluated the localization of Nicastrin, a γ-secretase subunit, at nanometer sized functional zones of the synapse. With the help of super resolution microscopy, we confirm that Nicastrin is organized into nanodomains of high molecular density within an excitatory synapse. A similar nanoorganization was also observed for APP and the catalytic subunit of γ-secretase, Presenilin 1, that were discretely associated with Nicastrin nanodomains. Though Nicastrin is a functional subunit of γ-secretase, the Nicastrin and Presenilin1 nanodomains were either colocalized or localized independent of each other. The Nicastrin and Presenilin domains highlight a potential independent regulation of these molecules different from their canonical secretase function. The collisions between secretases and substrate molecules decide the probability and rate of product formation for transmembrane proteolysis. Our observations of secretase nanodomains indicate a spatial difference in the confinement of substrate and secretases, affecting the local probability of product formation by increasing their molecular availability, resulting in differential generation of proteoforms even within single synapses. [ABSTRACT FROM AUTHOR]

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

γ-secretase is an intramembrane protease complex that catalyzes the proteolytic cleavage of amyloid precursor protein and Notch. Impaired γ-secretase function is associated with the development of Alzheimer's disease and familial acne inversa in humans. In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for defects in adaptive immunity, we identified animals within a single pedigree exhibiting both hypopigmentation of the fur and diminished T cell-independent (TI) antibody responses. The causative mutation was in Ncstn, an essential gene encoding the protein nicastrin (NCSTN), a member of the γ-secretase complex that functions to recruit substrates for proteolysis. The missense mutation severely limits the glycosylation of NCSTN to its mature form and impairs the integrity of the γ-secretase complex as well as its catalytic activity toward its substrate Notch, a critical regulator of B cell and T cell development. Strikingly, however, this missense mutation affects B cell development but not thymocyte or T cell development. The Ncstn allele uncovered in these studies reveals an essential requirement for NCSTN during the type 2 transitionalmarginal zone precursor stage and peritoneal B-1 B cell development, the TI antibody response, fur pigmentation, and intestinal homeostasis in mice. [ABSTRACT FROM AUTHOR]

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.

Background: Familial Hidradenitis Suppurativa and Familial Alzheimer's Disease are both associated with Gamma-Secretase Complex mutations; however, the two diseases are not epidemiologically associated. Understanding the molecular differences between the two diseases may aid in the development of hypotheses for differing pathogenesis and ultimately, targets for detection.Aims: To characterize the in silico structural and functional alterations to the Gamma Secretase Complex in documented mutations in Familial Hidradenitis Suppurativa, along with comparison of downstream substrate recognition and cleavage.Methods:In silico analysis of publicly available genomic data, assessment of protein structure and binding affinity using Swiss-model and Dynamut was undertaken. Differential Expression was expressed using Log Fold Change using the general framework for linear models in R. Differentially expressed genes (DEGs) were defined by FCH ≥1.5 or ≤−1.5 and false discovery rate (FDR ≤ 0.05).Results: Twenty three of 39 mutations in HS are degraded via nonsense mediated decay with altered substrate and binding affinity of substrates identified in the remaining mutations. Significant differential expression of ErbB4, SCNB1, and Tie1 in lesional skin was specific to Hidradenitis Suppurativa and EphB2, EPHB4, KCNE1, LRP6, MUSK, SDC3, Sortilin1 in blood specific to Familial Alzheimer's Disease.Discussion and Conclusions: We present the first in silico evidence as to the impact of documented mutations in Familial Hidradenitis Suppurativa. We also demonstrate unique substrate recognition and cleavage between Hidradenitis Suppurativa and Familial Alzheimer's Disease, providing a potential explanation as to why the two diseases do not occur within the same pedigree. These proteomic signatures may be a first step in identifying reliable biomarkers for Familial Hidradenitis Suppurativa.

γ-secretase, an intramembrane-cleaving aspartyl protease is involved in the cleavage of a large number of intramembrane proteins. The most prominent substrate is the amyloid precursor protein, whose proteolytic processing leads to the production of different amyloid Aβ peptides. These peptides are known to form toxic aggregates and may play a key role in Alzheimer's disease (AD). Recently, the three-dimensional structure of γ-secretase has been determined via Cryo-EM, elucidating the spatial geometry of this enzyme complex in different functional states. We have used molecular dynamics (MD) simulations to study the global dynamics and conformational transitions of γ-secretase, as well as the water and lipid distributions in and around the transmembrane domains in atomic detail. Simulations were performed on the full enzyme complex and on the membrane embedded parts alone. The simulations revealed global motions compatible with the experimental enzyme structures and indicated little dependence of the dynamics of the transmembrane domains on the soluble extracellular subunits. During the simulation on the membrane spanning part a transition between an inactive conformation (with catalytic residues far apart) toward a putatively active form (with catalytic residues in close proximity) has been observed. This conformational change is associated with a distinct rearrangement of transmembrane helices, a global compaction of the catalytically active presenilin subunit a change in the water structure near the active site and a rigidification of the protein fold. The observed conformational rearrangement allows the interpretation of the effect of several mutations on the activity of γ-secretase. A number of long-lived lipid binding sites could be identified on the membrane spanning surface of γ-secretase which may coincide with association regions of hydrophobic membrane helices to form putative substrate binding exosites.

Background: Alzheimer's disease (AD) is characterized by the deposition of amyloid-β peptide (Aβ) in the brain. Aβ is produced by sequential β- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting β- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aβ production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aβ generation without inhibiting any enzyme., Methods: An APP mutant mimicking secreted APPγ was overexpressed in cells to test β-cleavage and Aβ production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms., Results: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aβ. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aβ, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase., Conclusions: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aβ. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Background: The pathogenesis of hidradenitis suppurativa (HS) is not fully understood. This systematic review examined the latest evidence for molecular inflammatory pathways involved in HS as a chronic inflammatory skin disease.Methods: A systematic literature search was performed in PubMed/Medline and EMBASE from January 2013 through September 2017, according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA). Findings on HS pathogenesis were also compared with those of other immune-mediated inflammatory diseases (IMIDs) in a non-systematic review. In addition, current therapeutic options for HS are briefly discussed on the basis of the findings for the inflammatory pathways involved in HS.Results: A total of 32 eligible publications were identified by the systematic search; these were supplemented with three additional publications. The extracted data indicated that four key themes underlie the pathogenesis of HS and related syndromic conditions. First, nicastrin (NCSTN) and PSTPIP1 mutations are directly associated with auto-inflammatory disease. Secondly, the up-regulation of several cytokines including tumor necrosis factor-α and T helper-17/interleukin-23 are connected to auto-inflammatory mechanisms in the pathogenesis of HS. Thirdly, the microbiome of lesional skin differs significantly vs. normal-appearing skin. Fourthly, HS risk is enhanced through physiological and environmental factors such as smoking, obesity, and mechanical friction. There is significant overlap between the pathogenesis of HS, its syndromic forms and other IMIDs, particularly with respect to aberrations in the innate immune response.Conclusions: The evidence presented in this review supports HS as an auto-inflammatory skin disorder associated with alterations in the innate immune system. Based on these most recent data, an integrative viewpoint is presented on the pathogenesis of HS. Current management strategies on HS consist of anti-inflammatory therapies, surgical removal of chronic lesions, and lifestyle changes such as smoking cessation and weight loss. As large gaps remain in the understanding of the pathogenesis of HS, further research is warranted to ultimately improve the management and treatment of patients with HS and related syndromic conditions.

Triple negative breast cancer (TNBC) is observed in ~15% of breast cancers and results in poor survival and increased distant metastases. Within the tumor are present a small portion of cancer stem cells that drive tumorigenesis and metastasis. In this study, we aimed to elucidate whether the two natural compounds, celastrol and triptolide, inhibit stemness in TNBC. MDA-MB-231, BT20, and a patient-derived primary cells (PD-TNBC) were used in the study. Mammosphere assay was performed to assess the stemness. Both celastrol and triptolide treatment suppressed mammosphere formation. Furthermore, the compound suppressed expression of cancer stem cell marker proteins DCLK1, ALDH1, and CD133. Notch signaling plays a critical role in stem cells renewal. Both celastrol or triptolide reduced Notch -1 activation and expression of its downstream target proteins HES-1 and HEY-1. However, when NICD 1 was ectopically overexpressed in the cells, it partially rescued proliferation and mammosphere formation of the cells, supporting the role of notch signaling. Together, these data demonstrate that targeting stem cells and the notch signaling pathway may be an effective strategy for curtailing TNBC progression.

Abstract Biological sex exerts distinct influences on brain levels of the β-amyloid (Aβ) peptide in both clinical depression and Alzheimer disease (AD), yet studies in animal models focus primarily on males. We examined behavioral 'despair'/depression (using the tail-suspension test) and memory (using the novel object recognition task) in J20 (hAPP Swe/Ind) mice. Three month-old male (but not female) J20 mice exhibited less despair-like behavior, but more evidence of cognitive deficits. In young J20 mice, only soluble Aβ peptides –primarily Aβ(1–40)– were detected. There was no evidence of an effect on despair-like behavior in the six month-old J20 mice, although cognitive deficits were now evident in both sexes, and coincided with a greater proportion of the neurotoxic Aβ(1–42) species (in soluble as well as insoluble fractions). This age-dependent shift in Aβ peptide profile coincided with reduced expression of glycosylated species of ADAM-10 (α-secretase) and BACE1 (β-secretase), and an increased co-immunoprecipitation of presenilin-1 with nicastrin (components of the γ-secretase complex). Sex-dependent changes in depression-related monoaminergic, e.g. serotonin and dopamine (but not noradrenaline), systems were evident already in young J20 mice. It is critical to acknowledge that sex-dependent APP-related phenotypes might differentially influence modifiable depression-related monoaminergic signalling at some of the earliest pathological stages of clinical AD. Highlights • Sex-specific biochemical and behavioral phenotypes are observed in young hAPP mice. • Depression-like behavior does not align with cognitive deficits, particularly in very young, female hAPP mice. • Sex-specific strategies might have to be considered for clinical interventions in depression and/or Alzheimer disease. [ABSTRACT FROM AUTHOR]

Acne inversa (AI) is a chronic inflammatory disease of hair follicles. The pathogenesis of AI remains unclear. Haploinsufficiency of genes encoding γ-secretase components is the genetic basis for a subset of familial AI. Idiopathic guttate hypomelanosis (IGH) is a leukoderma characterized by multiple porcelain-white macules. Familial AI associated with IGH has not been reported previously. Herein, we present the pathogenic variation in a Chinese Han family with AI and IGH. Peripheral blood samples were collected from 16 members of the entire family. Eighteen exons and flanking introns of the NCSTN gene were amplified by polymerase chain reaction. Two hundred unrelated healthy Chinese subjects were used as controls. Sequencing results were analysed using CodonCode Aligner Software. Seven of the 16 family members in three generations were AI patients. Six AI patients also had IGH, while the other only had AI. One had IGH without AI. All AI patients carried the mutation, c.218delC, located in exon 4 of NCSTN. The deletion mutation led to a reading frame shift and the appearance of a premature termination codon (p.P73Lfs*15), resulting in the production of truncated protein. Family members without AI did not carry this mutation, indicating that it cosegregated with the phenotype. The mutation was not detected among the controls. This mutation has not been reported in the EXaC, HGMD, and dbSNP databases. In addition, we performed whole-exome sequencing on the proband and finally screened six candidate genes, ADAMTS2, BUB1B, CRB2, FBLN1, SEC24B, and WNK1, which we further validated in effected family members, and none of them were cosegregated. In conclusion, we identified a novel deletion mutation in exon 4 of NCSTN, which may underlie the molecular pathogenesis in this AI family. However, we found no relationship between this mutation and IGH. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by cognitive impairment. The neuropathological features of AD are the aggregation of extracellular amyloid β-protein (Aβ) and tau phosphorylation. Recently, AD was found to be associated with magnesium ion (Mg2+) deficit and tumor necrosis factor-alpha (TNF-α) elevation in the serum or brains of AD patients. To study the relationship between Mg2+ and TNF-α, we used human- or mouse-derived glial and neuronal cell lines or APP/PS1 transgenic (Tg) mice as in vitro and in vivo experimental models, respectively. Our data demonstrates that magnesium-L-threonate (MgT) can decrease the expression of TNF-α by restoring the levels of Mg2+ in glial cells. In addition, PI3-K/AKT and NF-κB signals play critical roles in mediating the effects of Mg2+ on suppressing the expression of TNF-α. In neurons, Mg2+ elevation showed similar suppressive effects on the expression of presenilin enhancer 2 (PEN2) and nicastrin (NCT) through a PI3-K/AKT and NF-κB-dependent mechanism. As the major components of γ-secretase, overexpression of presenilin 1 (PS1), PEN2 and NCT potentially promote the synthesis of Aβ, which in turn activates TNF-α in glial cells. Reciprocally, TNF-α stimulates the expression of PEN2 and NCT in neurons. The crosstalk between TNF-α and Aβ in glial cells and neurons could ultimately aggravate the development and progression of AD.

γ-Secretase complex, the assembly of nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (PEN-2) and Anterior pharynx defective 1 (Aph-1), catalyzes the cleavage of amyloid precursor protein to generate amyloid-β protein (Aβ), the main culprit of Alzheimer's disease. NCT becomes matured through complex glycosylation and play important role in γ-secretase activity by interacting with catalytic subunit PS. However, the role of NCT glycosylation on γ-secretase activity and substrate specificity is still unknown. The purpose of this study is to investigate the effect of NCT glycosylation on γ-secretase activity and substrate specificity in a group of glycosylation mutant lectin resistant CHO (Lec) cells. CHO Lec-1 cells lack glycosyltransferase-I, GnT-I, thus N -glycan on NCT are all oligomannose type, whereas CHO Lec-2 cells synthesize NCT containing sialic acid deficient oligosaccharides due to the impairment of cytidine 5′-monophosphate-sialic acid transporter. Here, we reported that mutant CHO Lec-1 and Lec-2 reduced γ-secretase activity in both cell-based and biochemical assays, and that CHO Lec-1 preferentially reduced Aβ generation. Endogenous level of γ-secretase complex, subcellular distribution of γ-secretase subunits and the level of functional γ-secretase complex remained unchanged in mutants. Interestingly, Coimmunoprecipitation study revealed that mutant γ-secretase could recognize substrate as well as parental γ-secretase. Our data suggests that thorough glycosylation of NCT is critical for enzymatic activity and substrate preference of γ-secretase. [ABSTRACT FROM AUTHOR]

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99- residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2, which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer's disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of theWT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aβ production. Each of these γ-secretase variants forms a stable oligomer with theWT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3- cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer's disease is critically evaluated. [ABSTRACT FROM AUTHOR]

Our recent study indicated that vitamin D and its receptors are important parts of the amyloid processing pathway in neurons. Yet the role of vitamin D receptor (VDR) in amyloid pathogenesis is complex and all regulations over the production of amyloid beta cannot be explained solely with the transcriptional regulatory properties of VDR. Given that we hypothesized that VDR might exist on the neuronal plasma membrane in close proximity with amyloid precursor protein (APP) and secretase complexes. The present study primarily focused on the localization of VDR in neurons and its interaction with amyloid pathology-related proteins. The localization of VDR on neuronal membranes and its co-localization with target proteins were investigated with cell surface staining followed by immunofluorescence labelling. The FpClass was used for protein-protein interaction prediction. Our results demonstrated the localization of VDR on the neuronal plasma membrane and the co-localization of VDR and APP or ADAM10 or Nicastrin and limited co-localization of VDR and PS1. E-cadherin interaction with APP or the γ-secretase complex may involve NOTCH1, NUMB, or FHL2, according to FpClass. This suggested complex might also include VDR, which greatly contributes to Ca+2 hemostasis with its ligand vitamin D. Consequently, we suggested that VDR might be a member of this complex also with its own non-genomic action and that it can regulate the APP processing pathway in this way in neurons. [ABSTRACT FROM AUTHOR]

Mutations in the Presenilin genes are the major genetic cause of Alzheimer's disease. Presenilin and Nicastrin are essential components of γ-secretase, a multi-subunit protease that cleaves Type I transmembrane proteins. Genetic studies in mice previously demonstrated that conditional inactivation of Presenilin or Nicastrin in excitatory neurons of the postnatal forebrain results in memory deficits, synaptic impairment, and age-dependent neurodegeneration. The roles of Drosophila Presenilin (Psn) and Nicastrin (Nct) in the adult fly brain, however, are unknown. To knockdown (KD) Psn or Nct selectively in neurons of the adult brain, we generated multiple shRNA lines. Using a ubiquitous driver, these shRNA lines resulted in 80-90% reduction of mRNA and pupal lethality--a phenotype that is shared with Psn and Nct mutants carrying nonsense mutations. Furthermore, expression of these shRNAs in the wing disc caused notching wing phenotypes, which are also shared with Psn and Nct mutants. Similar to Nct, neuron-specific Psn KD using two independent shRNA lines led to early mortality and rough eye phenotypes, which were rescued by a fly Psn transgene. Interestingly, conditional KD (cKD) of Psn or Nct in adult neurons using the elav-Gal4 and tubulin-Gal80ts system caused shortened lifespan, climbing defects, increases in apoptosis, and age-dependent neurodegeneration. Together, these findings demonstrate that, similar to their mammalian counterparts, Drosophila Psn and Nct are required for neuronal survival during aging and normal lifespan, highlighting an evolutionarily conserved role of Presenilin in neuronal protection in the aging brain. [ABSTRACT FROM AUTHOR]

Presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer-2 (Pen-2) have been considered the minimal essential subunits required to form an active γ-secretase complex. Besides PS, which has been widely believed to function as the catalytic subunit of the complex, the functional roles of the other subunits in the γ-secretase complex remain debatable. In the current study, we set out to determine the role of Pen-2 in γ-secretase activity. To this end, using knockout cells in combination with siRNA and immunoprecipitation approaches, our results revealed that Pen-2 together with presenilin are sufficient to form a functionally active enzyme to process Notch. Specifically, our data demonstrated that Pen-2 plays a crucial role in substrate binding, a mechanism by which Pen-2 contributes directly to the catalytic mechanism of γ-secretase activity. Our data also suggested that there may be different requirements for components to process AβPP and Notch. This information would be important for therapeutic strategy aimed at inhibition or modulation of γ-secretase activity. [ABSTRACT FROM AUTHOR]

Although Alzheimer's disease is a brain disorder, a number of peripheral alterations have been found in these patients; however, little is known about how the key genes involved in the pathophysiology express in peripheral cells such as lymphocytes during normal compared to neuropathological ageing. We analysed the expression of tau, of the amyloid precursor protein, of nicastrin and of the β-site APP cleaving enzyme genes by RT-PCR in lymphocytes from a small group of late-onset Alzheimer's disease patients, from aged patients suffering from neuropsychological conditions different from Alzheimer's and from cognitively healthy subjects divided in four groups by age. We also investigated correlations between gene expression and levels of blood pressure, glucose, total cholesterol and triglycerides as risk factors for Alzheimer's. Results show no tau expression in lymphocytes, a lack of detection of nicastrin expression in Alzheimer's patients and correlations between the medical conditions studied and gene expression in lymphocytes. We believe nicastrin gene expression in lymphocytes should be considered of interest for further analyses in a wider population to investigate whether it might represent a potential biomarker to differentiate Alzheimer's from other neuropsychological disorders.

The muscle-fiber phenotype of sporadic inclusion-body myositis (s-IBM), the most common muscle disease associated with aging, shares several pathological abnormalities with Alzheimer disease (AD) brain, including accumulation of amyloid-β 42 (Aβ42) and its cytotoxic oligomers. The exact mechanisms leading to Aβ42 production within s-IBM muscle fibers are not known.Aβ42 and Aβ40 are generated after the amyloid-precursor protein (AβPP) is cleaved by β-secretase and the γ-secretase complex. Aβ42 is considered more cytotoxic than Aβ40, and it has a higher propensity to oligomerize, form amyloid fibrils, and aggregate. Recently, we have demonstrated in cultured human muscle fibers that experimental inhibition of lysosomal enzyme activities leads to Aβ42 oligomerization.In s-IBM muscle, we here demonstrate prominent abnormalities of the γ-secretase complex, as evidenced by: a) increase of γ-secretase components, namely active presenilin 1, presenilin enhancer 2, nicastrin, and presence of its mature, glycosylated form; b) increase of mRNAs of these γ-secretase components; c) increase of γ-secretase activity; d) presence of an active form of a newly-discovered γ-secretase activating protein (GSAP); and e) increase of GSAP mRNA. Furthermore, we demonstrate that experimental inhibition of lysosomal autophagic enzymes in cultured human muscle fibers a) activates γ-secretase, and b) leads to posttranslational modifications of AβPP and increase of Aβ42. Since autophagy is impaired in biopsied s-IBM muscle, the same mechanism might be responsible for its having increased γ-secretase activity and Aβ42 production. Accordingly, improving lysosomal function might be a therapeutic strategy for s-IBM patients.

Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein β, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of protein-sphingolipid interactions within the membrane bilayer of living cells.

Hidradenitis suppurativa (HS) is a chronic, debilitating skin disease for which few treatment options are available. While most HS is sporadic, some rare kindred show a high-penetrance, autosomal-dominant inheritance. We wanted to identify rare variants that could contribute to HS risk in sporadic cases using candidate gene sequencing. We ultimately identified 21 genes for our capture panel. We included genes of the γ-secretase complex (n = 6) because rare variants in these genes sometimes cause familial HS. We added Notch receptor and ligand genes (n = 13) because γ-secretase is critical for processing Notch receptor signaling. Clinically, some people with PAPA (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, a rare inflammatory disease, have concurrent HS. Rare variants in PSTPIP1 are known to cause PAPA syndrome, so we included PSTPIP1 and PSTPIP2 in the capture panel. We screened 117 individuals with HS for rare variations and calculated the expected burden using Genome Aggregation Database (gnomAD) allele frequencies. We discovered two pathogenic loss-of-function variants in NCSTN . This class of NCSTN variant can cause familial HS. There was no increased burden of rare variations in any γ-secretase complex gene. We did find that individuals with HS had a significantly increased number of rare missense variants in the SH3 domain of PSTPIP1 . This finding, therefore, implicates PSTPIP1 variation in sporadic HS and further supports dysregulated immunity in HS. Our data also suggests that population-scale HS genetic research will yield valuable insights into disease pathology., Competing Interests: B. Kaffenberger has consulted for and received clinical trial funds from Novartis. B. Kirby has received clinical trial support from AbbVie, Almirall, Janssen, Merck Sharpe Dolme, Moonlake, Novartis, Pfizer, and UCB BioPharma; been a consultant for AbbVie, Almirall, Celgene, Janssen, Merck Sharpe Dolme, Moonlake, Novartis, Pfizer, and UCB; received honoraria from AbbVie, Alimrall, Celgene, Janssen, Lilly, Moonlake, Novartis, Pfizer, and UCB; and been on scientific advisory boards for AbbVie, Almirall, Celgene, Janssen, Lilly, Moonlake, Novartis, Pfizer, and UCB. D.J.M.-H. was a postdoctoral fellow during the research and writing of this paper but has since become an employee of Pfizer and holds stock in the company. J.K. has received clinical trial support from XBiotech and UCB BioPharma. M.A. has served as a consultant and/or advisor for ImClone, Bristol Myers Squibb, Astra Zeneca, Therakos, Aspire Bariatrics, Biogen, Amgen, Veloce, Adgero, Eli Lilly, Abbvie, UCB Biopharma, Innovaderm, Boehringer-Ingelheim, OnQuality, Novocure, Springworks, BioLinq, and Protagonist and has served as a principal investigator for InflamRx, Biogen, Astra Zeneca, Novartis, Boehringer-Ingelheim, Lutris, OnQuality, UCB BioPharma, InflamRx, Eli Lilly, InCyte, Abbvie, Moonlake, AnaptysBio, Hana Biosciences, Xoma, Moonlake, Moberg, Veloce, Biogen, Xbiotech, and Chemocentryx., (© 2023 The Authors.)

The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid β peptide (Aβ) with a higher Aβ42+Aβ43-to-Aβ40 (Aβ42(43)/Aβ40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aβ42(43)/Aβ40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD. [ABSTRACT FROM AUTHOR]