Neurodegeneration in Alzheimer's disease: caspases and synaptic element interdependence

Extensive genetic, biochemical, and histological evidence has implicated the amyloid-β peptide (Aβ) in Alzheimer's disease pathogenesis, and several mechanisms have been suggested, such as metal binding, reactive oxygen species production, and membrane pore formation. However, recent evidence argues for an additional role for signaling mediated by the amyloid precursor protein, APP, in part via the caspase cleavage of APP at aspartate 664. Here we review the effects and implications of this cleavage event, and propose a model of Alzheimer's disease that focuses on the critical nature of this cleavage and its downstream effects

Improved prediction of critical residues for protein function based on network and phylogenetic analyses

BACKGROUND: Phylogenetic approaches are commonly used to predict which amino acid residues are critical to the function of a given protein. However, such approaches display inherent limitations, such as the requirement for identification of multiple homologues of the protein under consideration. Therefore, complementary or alternative approaches for the prediction of critical residues would be desirable. Network analyses have been used in the modelling of many complex biological systems, but only very recently have they been used to predict critical residues from a protein's three-dimensional structure. Here we compare a couple of phylogenetic approaches to several different network-based methods for the prediction of critical residues, and show that a combination of one phylogenetic method and one network-based method is superior to other methods previously employed. RESULTS: We associate a network with each member of a set of proteins for which the three-dimensional structure is known and the critical residues have been previously determined experimentally. We show that several network-based centrality measurements (connectivity, 2-connectivity, closeness centrality, betweenness and cluster coefficient) accurately detect residues critical for the protein's function. Phylogenetic approaches render predictions as reliable as the network-based measurements, although, interestingly, the two general approaches tend to predict different sets of critical residues. Hence we propose a hybrid method that is composed of one network-based calculation – the closeness centrality – and one phylogenetic approach – the Conseq server. This hybrid approach predicts critical residues more accurately than the other methods tested here. CONCLUSION: We show that network analysis can be used to improve the prediction of amino acids critical for protein function, when utilized in combination with phylogenetic approaches. It is proposed that such improvement is due to the complementary nature of these approaches: network-based methods tend to predict as critical those residues that are highly connected and internal (i.e., non-surface), although some surface residues are indeed identified as critical by network analyses; whereas residues chosen by phylogenetic approaches display a lower overall probability of being surface inaccessible

A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer's disease mouse model.

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo, A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer's disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels

A Novel Motif Identified in Dependence Receptors

Programmed cell death signaling is a critical feature of development, cellular turnover, oncogenesis, and neurodegeneration, among other processes. Such signaling may be transduced via specific receptors, either following ligand binding—to death receptors—or following the withdrawal of trophic ligands—from dependence receptors. Although dependence receptors display functional similarities, no common structural domains have been identified. Therefore, we employed the Multiple Expectation Maximization for Motif Elicitation and the Motif Alignment and Search Tool software programs to identify a novel transmembrane motif, dubbed dependence-associated receptor transmembrane (DART) motif, that is common to all described dependence receptors. Of 3,465 human transmembrane proteins, 25 (0.7%) display the DART motif. The predicted secondary structure features an alpha helical structure, with an unusually high percentage of valine residues. At least four of the proteins undergo regulated intramembrane proteolysis. To date, we have not identified a function for this putative domain. We speculate that the DART motif may be involved in protein processing, interaction with other proteins or lipids, or homomultimerization

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75NTR and their effect on NF-κB activation

AbstractThe common neurotrophin receptor p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75NTR cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75NTR. Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75NTR at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-κB activation and tamoxifen-induced apoptosis in 293T cells expressing p75NTR. The results revealed that TRAF6-mediated NF-κB activation was suppressed by p75NTR and that the p75NTR-mediated NF-κB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75NTR intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75NTR-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75NTR-mediated signal transduction by physiological interaction through its third PDZ domain

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease

Human Embryonic Stem Cells Express Elevated Levels of Multiple Pro-Apoptotic BCL-2 Family Members

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms

The PDZ Domain as a Complex Adaptive System

Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the best-studied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by target-assisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinity-ranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order sub-cellular organizations as synapses, cell junctions, and others – organizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork

Efficient Identification of Critical Residues Based Only on Protein Structure by Network Analysis

Despite the increasing number of published protein structures, and the fact that each protein's function relies on its three-dimensional structure, there is limited access to automatic programs used for the identification of critical residues from the protein structure, compared with those based on protein sequence. Here we present a new algorithm based on network analysis applied exclusively on protein structures to identify critical residues. Our results show that this method identifies critical residues for protein function with high reliability and improves automatic sequence-based approaches and previous network-based approaches. The reliability of the method depends on the conformational diversity screened for the protein of interest. We have designed a web site to give access to this software at http://bis.ifc.unam.mx/jamming/. In summary, a new method is presented that relates critical residues for protein function with the most traversed residues in networks derived from protein structures. A unique feature of the method is the inclusion of the conformational diversity of proteins in the prediction, thus reproducing a basic feature of the structure/function relationship of proteins