Nuria Assa-Munt

The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis

The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, re-establishment of DCC expression suppresses tumorigenicity,. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.

Some insights into protein structural class prediction

It has been quite clear that the success rate for predicting protein structural class can be improved significantly by using the algorithms that incorporate the coupling effect among different amino acid components of a protein. However, there is still a lot of confusion in understanding the relationship of these advanced algorithms, such as the least Mahalanobis distance algorithm, the component‐coupled algorithm, and the Bayes decision rule. In this communication, a simple, rigorous derivation is provided to prove that the Bayes decision rule introduced recently for protein structural class prediction is completely the same as the earlier component‐coupled algorithm. Meanwhile, it is also very clear from the derivative equations that the least Mahalanobis distance algorithm is an approximation of the component‐coupled algorithm, also named as the covariant‐discriminant algorithm introduced by Chou and Elrod in protein subcellular location prediction (Protein Engineering, 1999; 12:107–118). Clarification of the confusion will help use these powerful algorithms effectively and correctly interpret the results obtained by them, so as to conduce to the further development not only in the structural prediction area, but in some other relevant areas in protein science as well. Proteins 2001;44:57–59.

Analysis of Apoptosis Protein Expression in Early-Stage Colorectal Cancer Suggests Opportunities for New Prognostic Biomarkers

Purpose: Although most stage II colon cancers are potentially curable by surgery alone, ∼20% of patients relapse, suggesting a need for establishing prognostic markers that can identify patients who may benefit from adjuvant chemotherapy. We tested the hypothesis that differences in expression of apoptosis-regulating proteins account for differences in clinical outcome among patients with early-stage colorectal cancer. Experimental Design: Tissue microarray technology was employed to assay the expression of apoptosis-regulating proteins by immunohistochemistry in 106 archival stage II colorectal cancers, making correlations with disease-specific survival. The influence of microsatellite instability (MSI), tumor location (left versus right side), patient age, and gender was also examined. Results: Elevated expression of several apoptosis regulators significantly correlated with either shorter (cIAP2; TUCAN) or longer (Apaf1; Bcl-2) overall survival in univariate and multivariate analyses. These biomarkers retained prognostic significance when adjusting for MSI, tumor location, patient age, and gender. Moreover, certain combinations of apoptosis biomarkers were highly predictive of death risk from cancer. For example, 97% of patients with favorable tumor phenotype of cIAP2low plus TUCANlow were alive at 5 years compared with 60% of other patients (P = 0.00003). In contrast, only 37% of patients with adverse biomarkers (Apaf1low plus TUCANhigh) survived compared with 83% of others at 5 years after diagnosis (P< 0.0001). Conclusions: Immunohistochemical assays directed at detection of certain combinations of apoptosis proteins may provide prognostic information for patients with early-stage colorectal cancer, and therefore could help to identify patients who might benefit from adjuvant chemotherapy or who should be spared it.

Dimerization‐dependent block of the proapoptotic effect of P75NTR

The biochemical mechanism by which neurons become dependent on neurotrophins for survival is unknown. We found previously that the common neurotrophin receptor, p75NTR, is a mediator of neurotrophin dependence and that this effect requires a novel type of domain dubbed a neurotrophin dependence domain. We report here that, in contrast to other proapoptotic receptors such as Fas, apoptosis induction by p75NTR requires monomerization, with dimerization inhibiting the effect. Blocking the proapoptotic effect of the monomer by dimerization requires a distinct domain that lies at the carboxyterminus of p75NTR. These results define a novel type of domain required for inhibiting apoptosis induction by p75NTR. J. Neurosci. Res. 60:587–593, 2000

Neurotrophin dependence domain

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.

Identification of a PU.1–IRF4 protein interaction surface predicted by chemical exchange line broadening

Relaxation values reflecting residue-specific line broadening revealed amino acids in the DNA-binding domain of PU.1 on a surface potentially involved in protein–protein interactions. Mutation of these amino acids did not cause protein unfolding but destabilized PU.1–DNA binding. Addition of IFN response factor 4 to form the ternary complex recovered binding stability. Fluorescence quenching experiments proved that this surface of PU.1 interacts with IFN response factor 4 during binding. Our results provide evidence that residues that display increased conformational exchange can be used to predict areas of protein–protein interactions.

A cytoplasmic peptide of the neurotrophin receptor p75NTR: induction of apoptosis and NMR determined helical conformation

The neurotrophin receptor (NTR) and tumor necrosis factor receptor family of receptors regulate apoptotic cell death during development and in adult tissues [Beutler and van Huffel, Science 264 (1994) 667–668]. We have examined a fragment of p75NTR from the carboxyl terminus of the receptor and a variant form of this peptide via NMR techniques and in vitro assays for apoptotic activity. The wild type peptide induces apoptosis and adopts a helical conformation oriented parallel to the surface of lipid micelles, whereas the variant form adopts a non‐helical conformation in the presence of lipid and shows no activity. These experiments suggest a link between structure and function of the two peptides.

Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3.

Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca2+ and Mg2+ bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin αIIbβ3. The library was used to select for peptides that bind to the integrin αIIbβ3 preferentially in Ca2+ versus Mg2+. Peptides were identified which bound selectively in each ion. The Ca2+-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg2+-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg2+-selected peptides contained a unique basic patch that protrudes from the base of the turn.