Sirt1 protects from K‐Ras‐driven lung carcinogenesis

The NAD⁺‐dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non‐small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K‐RAS. Therefore, we investigated the effect of SIRT1 on K‐RAS‐driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K‐RAS in a MEK and PI3K‐dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K‐Ras^G12V‐driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1‐Tg pneumocytes, isolated shortly after K‐Ras^G12V activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K‐RAS‐driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1–K‐RAS axis could be a therapeutic target for NSCLCs.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

Mitochondria and senescence: new actors for an old play

Mitochondrial dysfunction has been linked to both cellular senescence and ageing. Despite the relationship, it is still unclear whether mitochondria have a causal role in senescence. In this issue of The EMBO Journal, Correia‐Melo et al ( 2016 ) combine targeted depletion of mitochondria with impairment of their biogenesis to demonstrate that decreased numbers of mitochondria impair the senescence response. Their results suggest that targeting mitochondria could reduce the detrimental effects of senescence during ageing.     

Sequential 1H-NMR assignment and solution structure of bovine pancreatic ribonuclease A

Assignments for 1H-NMR resonances of most of the residues of bovine pancreatic ribonuclease (RNase A) have been obtained by sequence-specific methods. Identification and classification of spin systems have been carried out by two-dimensional phase-sensitive correlated spectroscopy (360 MHz) and single relayed coherence transfer spectroscopy. Sequence-specific assignments have been achieved by phase-sensitive two-dimensional nuclear Overhauser effect spectroscopy. To overcome the problem of spectral overlap use has been made of (a) an exhaustive analysis of partly exchanged RNase A (spectra in D2O), (b) a comparison with the subtilisin-modified enzyme (RNase S) and (c) small spectral perturbations caused by changes in pH and temperature. The secondary structure elements have been identified from the observed sequential, medium and long-range nuclear Overhauser effects together with data from amide-exchange rates. All information collected leads to the conclusion that the crystal and the solution structures are closely similar.     

Regulation of glycolysis in sea bass liver: phosphofructokinase isozymes

Sea bass (Dicentrarchus labrax L.) liver phosphofructokinase (PFK) presents biphasic kinetics with respect to fructose-6-phosphate (F-6-P) in experiments carried out with crude extract. After the enzyme had been purified, two isozymes have been detected after chromatographic treatment. The two isozymes present different kinetic behaviour PFK-L1, the first eluted phosphofructokinase activity shows positive cooperativity with respect to fructose-6-phosphate and PFK-L2, the second activity fraction, has a Hill coefficient of 0.38 (negative cooperativity). The first isozyme shows less affinity for fructose-6-phosphate than that shown by PFK-L2. The joint kinetics of both isozymes produces a biphasic kinetics with respect to fructose-6-phosphate, similar to that observed in crude extracts.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.