Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor.

Nicastrin is genetically linked to Notch/lin-12 signaling in C. elegans and is part of a large multiprotein complex along with Presenilin. Here we describe the isolation and characterization of Drosophila Nicastrin (Nic) mutants. Nic mutants and tissue clones display characteristic Notch-like phenotypes. Genetic and inhibitor studies indicate a function for Nicastrin in the gamma-secretase step of Notch processing, similar to Presenilin. Further, Nicastrin is genetically required for signaling from membrane-anchored activated Notch. In the absence of Nicastrin, Presenilin is destabilized and mature C-terminal subunits are absent. Nicastrin might recruit gamma-secretase substrates into the proteolytic complex as a prerequisite for Presenilin maturation and active complex assembly.     

Glutamate receptor subunit 3 is modified by site-specific limited proteolysis including cleavage by gamma-secretase

Ionotropic glutamate receptor (GluR) expression and function is regulated through multiple pre- and post-translational mechanisms. We find that limited proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3 short forms that are glycosylated and found in association with other full-length GluRs in the mouse brain and cultured primary neurons. A combination of mutagenesis and transfection into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the membrane-localized re-entry loop at or near the leucine-glycine pair (amino acids 585-586, GluR3sbeta) and a second site within a proline-rich PEST-like sequence in the first cytoplasmic loop (Asp570-Pro571, GluR3salpha). Generation of the prominent GluR3salpha form was effectively abolished in the mutant, GluR3D570A, but inhibitors of lysosomes, the proteasome, caspases, or calpains had no effect. The possible impact of cleavage on receptor function was suggested when the co-expression of the GluR3P571Stop mutant (creating GluR3salpha) co-assembled with other GluR subunits and decreased receptor function in Xenopus oocytes. In transiently transfected HEK293 cells, co-expression of GluR3salpha alters the relative association between GluR1 and GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of GluR3salpha imparts lateral membrane mobility to GluR complexes. These results suggest that limited proteolysis is another post-translational mechanism through which functional diversity and specialization between closely related GluR subunits is accomplished.     

In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand

Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.     

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.     

Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins

Ligand binding to receptors of the LIN-12/Notch family causes at least two proteolytic cleavages: one between the extracellular and transmembrane domains, and the other within the transmembrane domain. The transmembrane cleavage depends on Presenilin, a protein also required for transmembrane cleavage of beta-APP. Here, we have assayed the substrate requirements for Presenilin-dependent processing of Notch and other type I transmembrane proteins in vivo. We find that the Presenilin-dependent cleavage does not depend critically on the recognition of particular sequences in these proteins but rather on the size of the extracellular domain: the smaller the size, the greater the efficiency of cleavage. Hence, Notch, beta-APP, and perhaps other proteins may be targeted for Presenilin-mediated transmembrane cleavage by upstream processing events that sever the extracellular domain from the rest of the protein.     

tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia colimazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA^Pro14 D-loop or within the tRNA^Lys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.     

SNAP-23 is a target for calpain cleavage in activated platelets

The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.     

COPI-mediated retrograde transport is required for efficient gamma-secretase cleavage of the amyloid precursor protein

Sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of beta-amyloid peptide, which is a key determinant in Alzheimer's disease. Since several putative locations for gamma-secretase cleavage have been identified along the secretory pathway, trafficking of APP may be of importance for beta-amyloid peptide production. Here we have studied the role of retrograde transport in APP processing. We found that APP interacts with the beta subunit of the coatomer protein I (COPI) complex, which is involved in retrograde transport. In line with a role of retrograde trafficking in APP transport, inhibition of COPI-dependent transport altered APP trafficking, decreased APP cell surface expression, and coincided with a profound reduction in gamma-secretase cleavage. These results suggest that COPI-dependent retrograde transport is important for APP processing and influences production of beta-amyloid peptide.     

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1

Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP). However, topological differences in cleavage resulting in amyloid beta-peptide (Abeta) or the Notch-1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N-terminal Notch-1 Abeta-like fragment (Nbeta). Analysis of Nbeta by MALDI-TOF MS revealed that Nbeta is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of betaAPP at position 40 and 42 of the Abeta domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer's disease-associated PS1 mutations similar to the pathological endoproteolysis of betaAPP. Considering these similarities between intramembranous processing of Notch and betaAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/gamma-secretase.     

Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

Background

The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown.

Results

Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin.

Conclusion

We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.     

The receptor for the orexigenic peptide melanin-concentrating hormone is a G-protein-coupled receptor

Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.     

Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by gamma-secretase

gamma-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/epsilon) and again in the middle of the TMD (at S4/gamma). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/epsilon site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only gamma-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/gamma) is dependent on the S3/epsilon cleavage. Notch (and perhaps all gamma-secretase substrates) may be cleaved by sequential proteolysis starting at S3.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.     

Functional PPAR-gamma receptor is a novel therapeutic target for ACTH-secreting pituitary adenomas

Adrenocorticotrophic hormone (ACTH)-secreting pituitary tumors are associated with high morbidity due to excess glucocorticoid production. No suitable drug therapies are currently available, and surgical excision is not invariably curative. Here we demonstrate immunoreactive expression of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) exclusively in normal ACTH-secreting human anterior pituitary cells: PPAR-gamma was abundantly expressed in all of six human ACTH-secreting pituitary tumors studied. PPAR-gamma activators induced G0/G1 cell-cycle arrest and apoptosis and suppressed ACTH secretion in human and murine corticotroph tumor cells. Development of murine corticotroph tumors, generated by subcutaneous injection of ACTH-secreting AtT20 cells, was prevented in four of five mice treated with the thiazolidinedione compound rosiglitazone, and ACTH and corticosterone secretion was suppressed in all treated mice. Based on these findings, thiazolidinediones may be an effective therapy for Cushing disease