I. Saira Mian

The premature ageing syndrome protein, WRN, is a 3'-->5' exonuclease.

Werner syndrome (WS) is a human autosomal recessive disorder that causes the premature appearance of a partial array of disorders characteristic of old age1,2. These disorders include atherosclerosis, cancer, type 2 diabetes, osteoporosis, cataracts, wrinkled skin and grey hair, among other ailments. Cells cultured from WS subjects have a shortened replicative life span3,4 and elevated rates of chromosome translocations, large deletions and homologous recombination5,6. The gene defective in WS, WRN, encodes a large RecQ-like DNA helicase7 of 1432 aa. Defects in another human RecQ-like helicase, BLM, result in Bloom’s syndrome8 (BS), a genetic disorder that is quite different from WS. BS is manifested by short stature, neoplasia, immunodeficiency and high risk of cancer. Cells from BS subjects show an increase in sister chromatid exchanges. DNA helicases can function in replication, repair, recombination, transcription or RNA processing. As WRN and BLM share no obvious homology outside the helicase domain, the non-helicase domains probably determine in which process each RecQ-like helicase participates, which provides the basis for the disparate cellular and organismal phenotypes that result from defects in these proteins. Statistical sequence analyses showed subtle but significant similarities between WRN and several 3′→5′ exonucleases9,10. To test the prediction that WRN is an exonuclease, we produced tagged recombinant wild-type and mutant WRN proteins. Two mutants had amino-acid substitutions at either position 82 (D82A) or 84 (E84A), two of the five residues predicted to be critical for exonuclease activity9,10. A third mutant had a substitution at position 577 (K577M), which abolished WRN helicase activity11. The fourth mutant was an N-terminal fragment (aa 1–333; N333) containing the putative exonuclease domain, but lacking the helicase domain. A tagged 36-aa vector-derived peptide served as a negative control (mock). Purified WRN and mock proteins were incubated with doubled-stranded DNA substrates. Wild-type WRN degraded a 5′ labelled substrate to a series of smaller, labelled products (Fig. 1a), and a 3′ labelled substrate to a single labelled product that migrated as a mononucleotide (Fig. 1b). Thus, WRN degraded DNA with 3′→5′ directionality. Although mock and full-length WRN preparations contained low levels of a contaminating 5′→3′ exonuclease, as shown by release of the 5′ label as a mononucleotide (Figs 1a,​,2b),2b), 3′→5′ degradation was entirely dependent on WRN. Fig. 1 Exonuclease activity of wild-type WRN and the N333 fragment. 6×his-tagged proteins were purified from baculovirus-infected insect cells using either nuclear (WRN, D82A, E84A, K577M, mock control) or cytosolic (N333, mock control) extracts. WRN, ... Fig. 2 Helicase and exonuclease activities of wild-type and mutant WRN proteins. a, WRN, K577M, E84A, D82A or mock proteins (10 ng) were assayed for helicase activity by incubating 5′ 32P-labelled DNA substrate (0.4 pmol; Fig. 1a) in helicase assay buffer ... WRN exonuclease activity resided in the N terminus. N333, which was essentially free of contaminating 5′→3′ exonuclease, degraded 5′ and 3′ labelled substrates similarly to full-length WRN (Fig. 1c,d). When incubated with a 374-bp DNA fragment labelled at the 3′ end with 32P, and internally with 3H, N333 released most of both labels (Fig. 1e). Thus, the WRN exonuclease is capable of substantial DNA degradation. Consistent with 3′→5′ directionality, N333 released 32P from 3′ ends more rapidly than 3H from internal residues. In addition, gel-purified N333, which lacked contaminating nuclease activities, efficiently removed the 3′, but not the 5′, label when incubated with DNA substrates labelled at either the 3′ or the 5′ end (Fig. 1f). Genetic evidence for WRN exonuclease activity was obtained by introducing point mutations at critical amino acids in the exonuclease domain (D82A and E84A). These mutants retained the wild-type level of helicase activity (Fig. 2a), but had little or no 3′→5′ exonuclease activity, using either a 5′ (Fig. 2b) or 3′ (Fig. 2c) 32P-labelled substrate, and were indistinguishable from mock protein in this regard (Fig. 2d). The K577M mutant, in contrast, was devoid of helicase activity (Fig. 2a), as expected, but had 3′→5′ exonuclease activity comparable to that of wild-type WRN (Fig. 2b–d). Our data indicate that WRN is indeed a 3′→5′ exonuclease. This activity resides in the N terminus, and is physically and functionally separable from the helicase activity. The identification of an exonuclease activity in WRN clearly distinguishes it from other human RecQ-like helicases, and may help explain the differences between WS and BS. What are the functions of the WRN exonuclease in vivo? It may participate in recombination and DNA repair. Exonucleases are integral components of some recombination pathways12, and WRN appears to have a role in recombination5,6,13. The finding that WS cells are hypersensitive to the DNA damaging agent 4-nitroquinoline-1-oxide14 suggests a role for WRN in DNA repair. Finally, WRN is homologous to FFA-1 (replication focus-forming activity 1) in Xenopus laevis15, raising the possibility that WRN may also be involved in DNA replication. In this context, the WRN exonuclease may provide 3′→5′ proofreading function to DNA polymerases that lack such activity. Whatever the case, an understanding of the functions of WRN exonuclease and their relationships to the other functions of WRN will lead to new insights into the molecular and cellular basis for WS and a subset of age-associated pathologies.

LMNA mutations in atypical Werner's syndrome

BACKGROUND Werners syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werners syndrome (wild-type WRN). METHODS Of 129 index patients referred to our international registry for molecular diagnosis of Werners syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werners syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS In four (15%) of 26 patients with atypical Werners syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werners syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION Our findings indicate that Werners syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Association of human aging with a functional variant of klotho

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-β-d-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.

mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: Genetic and biochemical evidence that Vhs is a nuclease

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.

Analysis of Telomerase Processivity: Mechanistic Similarity to HIV-1 Reverse Transcriptase and Role in Telomere Maintenance

The key protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT)-like domain that is conserved in enzymes encoded by retroviruses and retroelements. Structural and functional analysis of HIV-1 RT suggests that RT processivity is governed, in part, by the conserved motif C, motif E, and a C-terminal domain. Mutations in analogous regions of the yeast TERT were found to have anticipated effects on telomerase processivity in vitro, suggesting a great deal of mechanistic and structural similarity between TERT and retroviral RTs, and a similarity that goes beyond the homologous domain. A close correlation was uncovered between telomerase processivity and telomere length in vivo, suggesting that enzyme processivity is a limiting factor for telomere maintenance.

SATB1 Cleavage by Caspase 6 Disrupts PDZ Domain-Mediated Dimerization, Causing Detachment from Chromatin Early in T-Cell Apoptosis

ABSTRACT SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ∼50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

Statistical Modeling, Phylogenetic Analysis and Structure Prediction of a Protein Splicing Domain Common to Inteins and Hedgehog Proteins

Inteins, introns spliced at the protein level, and the hedgehog family of proteins involved in eucaryotic development both undergo autocatalytic proteolysis. Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis. The HMM characterizes the common features of this domain including the position where a site-specific DNA endonuclease domain is inserted in the majority of the inteins. The HMM was used to identify several new putative inteins, such as that in the Methanococcus jannaschii klbA protein, and to generate a multiple sequence alignment of sequences possessing this domain. Phylogenetic analysis suggests that hedgehog proteins evolved from inteins. Secondary and tertiary structure predictions suggest that the domain has a structure similar to a beta-sandwich. Similarities between the serine protease cleavage mechanism and the protein splicing reaction mechanism are discussed. Examination of the locations of inteins indicates that they are not inserted randomly in an extein, but are often inserted at functionally important positions in the host proteins. A specific and sensitive HMM for a domain present in klbA proteins identified several additional bacterial and archaeal family members, and analysis of the site of insertion of the intein suggests residues that may be functionally important. This domain may play a role in formation of surface-associated protein complexes.

The thorny path linking cellular senescence to organismal aging

Patil et al. The thorny path linking cellular senescence to organismal aging Christopher K. Patil , I. Saira Mian , Judith Campisi a a a,b, a Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 USA b Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94545 USA * Telephone: Email: jcampisi@lbl.gov Keywords: Cellular senescence; Telomeres; Tumor suppression; Stress resistance.

A Conserved Telomerase Motif within the Catalytic Domain of Telomerase Reverse Transcriptase Is Specifically Required for Repeat Addition Processivity

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition. The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B′, within the so-called fingers domain. Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain). Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance. Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence. Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA. This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition. Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.

SKI pathways inducing progression of human melanoma.

The proteins SKI and SnoN are implicated in processes as diverse as differentiation, transformation and tumor progression. Until recently, SKI was solely viewed as a nuclear protein with a principal function of inhibiting TGF-β signaling through its association with the Smad proteins. However, new studies suggest that SKI plays additional roles not only inside but also outside the nucleus. In normal melanocytes and primary non-invasive melanomas, SKI localizes predominantly in the nucleus, whereas in primary invasive melanomas SKI displays both nuclear and cytoplasmic localization. Intriguingly, metastatic melanoma tumors display nuclear and cytoplasmic or predominantly cytoplasmic SKI distribution. Cytoplasmic SKI is functional, as it associates with Smad3 and prevents its nuclear localization mediated by TGF-β. SKI can also function as a transcriptional activator, targeting the β -catenin pathway and activating MITF and NrCAM, two proteins involved in survival, migration and invasion. Intriguingly, SKI appears to live a dual life, one as a tumor suppressor and another as a transforming protein. Loss of one copy of mouse ski increases susceptibility to tumorigenesis in mice, whereas its overexpression is associated with cancer progression of human melanoma, esophageal, breast and colon. The molecular reasons for such dramatic change in SKI function appear to result from new acquired activities. In this review, we discuss the mechanisms by which SKI regulates crucial pathways involved in the progression of human malignant melanoma.