Defects in telomere maintenance molecules impair osteoblast differentiation and promote osteoporosis

Osteoporosis and the associated risk of fracture are major clinical challenges in the elderly. Telomeres shorten with age in most human tissues, including bone, and because telomere shortening is a cause of cellular replicative senescence or apoptosis in cultured cells, including mesenchymal stem cells (MSCs) and osteoblasts, it is hypothesized that telomere shortening contributes to the aging of bone. Osteoporosis is common in the Werner (Wrn) and dyskeratosis congenita premature aging syndromes, which are characterized by telomere dysfunction. One of the targets of the Wrn helicase is telomeric DNA, but the long telomeres and abundant telomerase in mice minimize the need for Wrn at telomeres, and thus Wrn knockout mice are relatively healthy. In a model of accelerated aging that combines the Wrn mutation with the shortened telomeres of telomerase (Terc) knockout mice, synthetic defects in proliferative tissues result. Here, we demonstrate that deficiencies in Wrn^−/– Terc^−/– mutant mice cause a low bone mass phenotype, and that age-related osteoporosis is the result of impaired osteoblast differentiation in the context of intact osteoclast differentiation. Further, MSCs from single and Wrn^−/– Terc^−/– double mutant mice have a reduced in vitro lifespan and display impaired osteogenic potential concomitant with characteristics of premature senescence. These data provide evidence that replicative aging of osteoblast precursors is an important mechanism of senile osteoporosis.     

Hydrophobic cluster analysis and classification of sixteen bacterial alginate lyases

Sixteen alginate lyases whose primary sequences have been reported were compared, and classified into the following three groups on the basis of the identity of their primary sequences. Strong homology (>50%): A-AlgL, A-AlgL*, P-AlgL, P-AlgL*, and AlgA; weak homology (>20%): ALY, AlxM, P-Aly, K-Aly, AlyPG, AlgVGI, AlgVGII, and AlgVGIII; little homology (<20%): ALYII, Al-III, and AlgVMI. Using hydrophobic cluster analysis (HCA), a secondary structure prediction method, the sixteen alginate lyases were placed into the following classes. Class 1: AlgA, A-AlgL, A-AlgL*, P-AlgL, and P-AlgL*; Class 2: AlgVMI and Al-III; Class 3: ALY and AlxM; Class 4A: ALYII, K-Aly, P-Aly, and AlyPG; Class 4B: AlgVGI and AlgVGII; Class 5: AlgVGIII, which is put in a class of its own, because it is unlike any of the other alginate lyases.     

Comparison of the hemocyanin beta-barrel with other Greek key beta-barrels: possible importance of the "beta-zipper" in protein structure and folding.

The Greek key beta-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key beta-barrel, which forms the core of the third domain of this protein. The hemocyanin beta-barrel was found to be structurally very similar to the beta-barrels of the immunoglobulin domains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumor proteins. The structural similarity within this group of protein families is not accompanied by an evolutionary or functional relationship. It is therefore possible to study structure-sequence relations without bias from nonstructural constraints. The present study reports a conserved pattern of features in these Greek key beta-barrels that is strongly suggestive of a folding nucleation site. This proposed nucleation site, which we call a "beta-zipper," shows a pattern of well-conserved, large hydrophobic residues on two sequential beta-strands joined by a short loop. Each beta-zipper strand is near the center of one of the beta-sheets, so that the two strands face each other from opposite sides of the barrel and interact through their hydrophobic side chains, rather than forming a hydrogen-bonded beta-hairpin. Other protein families with Greek key beta-barrels that do not as strongly resemble the immunoglobulin fold--such as the azurins, plastocyanins, crystallins, and prealbumins--also contain the beta-zipper pattern, which might therefore be a universal feature of Greek key beta-barrel proteins.     

Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis

A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.     

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow (BM) failure, and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X-linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1(Δ15) mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase-dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1(Δ15) mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N-acetyl cysteine (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1(Δ15) mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by NAC treatment. These results suggest that a pathogenic Dkc1 mutation accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X-linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.