Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit^-/- MEFs escape senescence to become immortal more rapidly than Fhit^+/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.     

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.     

The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression

The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.     

Assembly with the Cul4A-DDB1DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.     

Modifier effects between regulatory and protein-coding variation

Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis) is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs). We predict that about 18% (1,502 out of 8,233 nsSNPs) of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants.     

Antimicrobial activity of lysozyme isoforms: Key molecular features

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry‐heating increasing lysozyme activity. Dry‐heated lysozyme (DH‐L) is a mixture of isoforms (isoaspartyl, native‐like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH‐L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH‐L against E. coli , indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.     

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.     

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.