Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

Proteolytic processing of the amyloid precursor protein by ␤-secretase yields A4CT (C99), which is cleaved further by the as yet unknown ␥-secretase, yielding the ␤-amyloid (A␤) peptide with 40 (A␤ 40 ) or 42 residues (A␤ 42 ). Because the position of ␥-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the A␤ domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of ␥-secretase (A␤ 42 The main proteinaceous component of the amyloid plaques found in the brains of patients with Alzheimer's disease (AD) is ␤-amyloid (A␤; refs. 1 and 2), an Ϸ4-kDa peptide that is derived from the larger amyloid precursor protein (APP; ref.3). APP processing by the as yet unidentified protease activities, termed ␣-, ␤-, and ␥-secretases, leads to a variety of different soluble and membrane-bound proteins (for reviews, see refs. 4 and 5). The ␣-secretase activity cleaves APP within the A␤ domain and thus precludes the generation of A␤. This cleavage yields secretory ␣-APPs, comprising most of the N-terminal ectodomain of APP, and the remaining membrane-bound C-terminal fragment p3CT. Alternatively, APP can be cleaved by the ␤-secretase activity at the N terminus of A␤, generating a truncated, soluble ␤-APPs and a C-terminal fragment of 99 residues (A4CT, C99). The ␤-secretase product A4CT contains the entire A␤ domain, the transmembrane domain, and the cytoplasmic tail of APP and represents the direct precursor for A␤ (6, 7).Both membrane-bound C-terminal fragments of APP, A4CT and p3CT, are cleaved by the ␥-secretase activity within their transmembrane domains at the C terminus of A␤ or p3, thus releasing the 40-and 42-residue A␤ peptides (A␤ 40 and A␤ 42 ) and the 24-26 residue p3 peptides (p3 40 and p3 42 ) (8-11). Most cells secrete both peptides A␤ and p3 into the conditioned medium. In neuronal cells, as in primary hippocampal neurons and in kidney 293 cells, A␤, but not p3, also can be found intracellularly and does not seem to be secreted (12-16).The major A␤ species secreted by cultured cells expressing wild-type (wt) APP is A␤ 40 ; the minor species is A␤ 42 (17). Mutations in the APP close to the ␥-cleavage site have been shown to alter the cleavage specificity of the ␥-secretase activity (A␤ 42 ͞A␤ 40 ratio; refs. 14 and 18-20). However, the factors that determine this cleavage specificity are unknown. Experiments with inhibitors of ␥-secretase activity suggest that distinct proteases generate the A␤ 40 and A␤ 42 peptides (11,(21)(22)(23)), but it is not known whether these enzymes are related or not.Furthermore, although ␥-cleavage occurs in the transmembrane domain of A4CT, it is not clear whether the cleavage occurs while A4CT is inserted into the membrane or after release of A4CT from the membrane. Understanding the substrate specificity of the ␥-secretase activity is of great importance, because the cleavage at residue 42 ...

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPα), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPα degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPα. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPα, which account for their differential ability to induce leukemia.