Release Of Intracellular Domain Research Articles

Structural study of regulated intramembrane proteolysis of p75NTR by γ-secretase.

Programmed cell death (PCD) maintains the tissue size in a multicellular organism. Uncontrolled PCD in neurons has long been implicated in neurodegenerative diseases, e.g. Alzheimer's disease. p75 neurotrophin receptor (p75NTR) plays an important role in regulating neuronal growth and also involves in apoptotic signaling, which leads to morphological changes in cells in PCD. When binding to nerve growth factor (NGF), p75NTR maintains cell survival and proliferation; on the contrary, p75NTR induces apoptosis when the NGF precursor (pro-NGF) binds onto p75NTR. The complex formation of the pro-NGF, p75NTR, and sortilin initiates the apoptotic signaling. The subsequent proteolytic processing on p75NTR by γ-secretase relays the signaling to the neuronal apoptosis. p75NTR, the founding member of tumor necrosis factor (TNF) receptor superfamily, is a type I transmembrane protein, involved in the downstream cell death signaling after the release of intracellular domain (ICD) through regulated intramembrane proteolysis (RIP) executed by γ-secretase. Detailed molecular mechanism of how the γ-secretase cleaves p75NTR and releases its death domain is still unknown. To answer this question, our ultimate goal is to study the molecular mechanism of the p75NTR cleavage by γ-secretase using cryogenic electron microscopy (cryo-EM). We generated four p75NTR mutants with various truncated mutants in Sf9 insect cells to determine the cleavage site for the 𝛾-secretase. We characterized the bindings using size-exclusion chromatography, SDS-PAGE, and negative-stain EM. p75NTR mutants generated and characterized their bindings with γ -secretase. We will stabilize the complex formation and probe the interaction between these proteins using single-particle cryo-EM. The result will gain a fundamental understanding of how p75NTR cleavage triggers the neuronal apoptosis signaling.

Abstract P2-10-07: Ep-ICD overexpression associates with poor prognosis in invasive ductal carcinoma

Abstract Background: Despite improvements in treatment strategies recurrence rates are still high among breast cancer patients. This may be attributed to heterogeneous nature of breast cancers representing varied morphologic and biological features, behavior, and response to therapy. Even among breast tumors of similar histologic type and grade, prognosis varies. Currently, breast cancer prognosis assessment methods have limited accuracy, are expensive, and in 20-30% of cases lead to over-treatment with adverse effects. None of the currently known prognostic factors has the ability to predict accurately which breast cancer patients are at high risk of recurrence. Thus, there is an increasing need for identification and validation of prognostic markers for assessment of risk for disease recurrence in breast cancer patients. Epithelial cell adhesion molecule (EpCAM) is a glycosylated, 30- to 40-kDa type I membrane protein, expressed in several human epithelial tissues and overexpressed in cancers, as well as in progenitors, normal and cancer stem cells, and is implicated in epithelial mesenchymal transition (EMT). Regulated intra-membrane proteolysis (RIP) of EpCAM by tumor-necrosis-factor alpha converting enzyme (TACE) results in shedding of its extracellular domain (EpEx) and release of intracellular domain, Ep-ICD, into the cytoplasm. Ep-ICD can signal into the cell nucleus by engagement of components of the Wnt pathway proteins including four and one half LIM domains protein 2 (FHL2), β-catenin and Lef, leading to activation of its oncogenic activity. Objective. Evaluate the prognostic significance of Ep-ICD overexpression in invasive ductal carcinoma (IDC). Methodology: Formalin fixed paraffin embedded (FFPE) tissue sections obtained from IDCs (n = 180) and normal breast tissues (n = 45) were used for immunostaining for Ep-ICD using specific monoclonal antibody. A semi-quantitative visual scoring of the immunostaining results for Ep-ICD based on percentage of tumor cells stained and intensity of scoring was used to compare the expression in breast cancers and normal tissues. Statistical analysis was carried out to determine the association of Ep-ICD expression with clinical outcome. Results: Among the 180 IDCs analyzed, nuclear Ep-ICD was observed in 75 tissues (41.7%) while cytoplasmic positivity was observed in 145 tissues (80.6%). In comparison, nuclear Ep-ICD localization was observed only in 11 normal tissues (23.9%) and cytoplasmic positivity was observed in 39 normal tissues (86.7%). The nuclear / cytoplasmic Ep-ICD expression has been correlated with at least 5 years follow-up data of 180 breast cancer patients after primary treatment. Kaplan Meier survival analysis showed significantly reduced 5 year disease free survival in IDC patients showing nuclear positivity (p < 0.001) or cytoplasmic positivity (p = 0.048). In Cox multivariate regression analysis, nuclear Ep-ICD overexpression emerged as an independent indicator of poor prognosis in IDCs (p = 0.008, H.R. = 81.18). Conclusion: Among invasive ductal carcinomas of the breast, nuclear Ep-ICD overexpression predicts reduced 5-year disease free survival. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-10-07.

γ-Secretase-mediated Release of the Low Density Lipoprotein Receptor-related Protein 1B Intracellular Domain Suppresses Anchorage-independent Growth of Neuroglioma Cells

The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a gamma-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release.

Differential Glycosylation Regulates Processing of Lipoprotein Receptors by γ-Secretase

The low density lipoprotein (LDL) receptor-related protein 1 (LRP1) belongs to a growing number of cell surface proteins that undergo regulated proteolytic processing that culminates in the release of their intracellular domain (ICD) by the intramembranous protease gamma-secretase. Here we show that LRP1 is differentially glycosylated in a tissue-specific manner and that carbohydrate addition reduces proteolytic cleavage of the extracellular domain and, concomitantly, ICD release. The apolipoprotein E (apoE) receptor-2 (apoER2), another member of the LDL receptor family with functions in cellular signal transmission, also undergoes sequential proteolytic processing, resulting in intracellular domain release into the cytoplasm. The penultimate processing step also involves cleavage of the apoER2 extracellular domain. The rate at which this cleavage step occurs is determined by the glycosylation state of the receptor, which in turn is regulated by the alternative splicing of an exon encoding several O-linked sugar attachment sites. These findings suggest a role for differential and tissue-specific glycosylation as a physiological switch that modulates the diverse biological functions of these receptors in a cell-type specific manner.