1H, 13C and 15N resonance assignment of 6aJL2(R25G), a highly fibrillogenic λVI light chain variable domain

An allotypic variation at position 25 influences the fibrillogenicity of λVI light chains, which are related to humoral immune response and have been associated with AL amyloidosis. The full resonance assignment and a preliminary structural characterization of 6aJL2(R25G) are reported. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Single Mutation in Framework 2 of the Heavy Variable Domain Improves the Properties of a Diabody and a Related Single-Chain Antibody

Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single‐chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy‐chain variable domain. Diabody D4 displayed an increase in T_m (thermal transition midpoint temperature) of 6.3 °C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem. 286, 6143–6151]. The new variant, scFv LER, displayed an increase in T_m of 3.4 °C and was capable of neutralizing 2 LD₅₀ of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.     

Evolutionary Dynamics of ompA,the Gene Encoding the Chlamydia trachomatis Key Antigen

Chlamydia trachomatis is the trachoma agent and causes most bacterial sexually transmitted infections worldwide. Its major outer membrane protein (MOMP) is a well-known porin and adhesin and is the dominant antigen. So far, investigation of MOMP variability has been focused mainly on molecular epidemiological surveys. In contrast, we aimed to evaluate the impact of the host pressure on this key antigen by analyzing its evolutionary dynamics in 795 isolates from urogenital infections, taking into account the MOMP secondary structure and the sizes/positions of antigenic regions. One-third of the specimens showed a mutational drift from the corresponding genotype, where ∼42% of the mutations had never been described. Amino acid alterations were sixfold more frequent within B-cell epitopes than in the remaining protein (P = 0.027), and some mutations were also found within or close to T-cell antigenic clusters. Interestingly, the two most ecologically successful genotypes, E and F, showed a mutation rate 60.3-fold lower than that of the other genotypes (P < 10⁻⁸), suggesting that their efficacy may be the result of a better fitness in dealing with the host immune system rather than of specific virulence factors. Furthermore, the variability exhibited by some genetic variants involved residues that are known to play a critical role during the membrane mechanical movements, contributing to a more stable and flexible porin conformation, which suggests some plasticity to deal with environmental pressure. Globally, these MOMP mutational trends yielded no mosaic structures or important phylogenetic changes, but instead yielded point mutations on specific protein domains, which may enhance pathogen''s infectivity, persistence, and transmission.Chlamydia trachomatis is an obligate intracellular human pathogen that can be classified into 18 serovariants based on the immunoreactivity of its major outer membrane protein (MOMP) or ompA (which encodes MOMP) polymorphism: serovars A to C and Ba are responsible for trachoma; serovars D to K, Da, Ia, and Ja are normally associated with infection of the urogenital tract; and serovars L1 to L3 cause lymphogranuloma venereum (22). This preference for particular cell types is not exclusive, and therefore ocular strains can occasionally be found in the urogenital tract and vice versa. However, it is thought that only L1 to L3 strains possess the ability to invade the inguinal lymph nodes.MOMP has been implicated in the mechanisms of attachment, infection, and/or pathogenesis due to its variability, surface exposure, and antigenic properties. Previous studies have shown that MOMP may act as a putative cytoadhesin by promoting nonspecific interactions with host cells (64). This major chlamydial membrane component, which constitutes about 60% of the membrane dry weight (9), is also thought to play a role in maintaining structural integrity of the organism (9, 10) by forming a trimeric structure (66). Also, during chlamydial replication, MOMP may act as a porin (6) that is folded into a β-barrel structure containing five constant domains (CDI to CDV) of transmembrane β-strands and periplasmic turns and four highly variable surface-exposed domains (VDI to VDIV) (34, 59, 69). Furthermore, MOMP possesses species- and serovar-specific epitopes (2, 48, 73, 74) that are able to elicit both the humoral (B-cell mediated through the production of antibodies) and cellular (T-cell mediated and also influencing the B-cell response) immune responses, making this dominant chlamydial antigen a potential candidate for the development of vaccines and therapeutic strategies (8, 17, 23, 61). Indeed, although no efficacious chlamydial vaccine has been developed so far, the use of inactivated or live-attenuated pathogens has been replaced by peptide or subunit vaccines, and MOMP is definitely one of the leading candidates (19).To improve our knowledge of the effects of the host pressure on MOMP and also of the molecular epidemiology of the circulating C. trachomatis strains, it is imperative to investigate genetic variability in ompA. Here, we performed a sequence-based analysis of the ompA mutational trends in clinical isolates that were collected from patients with sexually transmitted C. trachomatis infections. So far, most studies have been limited to a small number of strains with variations in ompA (16, 33, 36, 42, 44, 47, 49, 52, 57, 62) or were restricted to the analysis of VDs (7, 14, 15, 32, 63), discarding the CDs, which contain numerous cytotoxic T lymphocyte (CTL) and T helper (Th) cell epitopes (30, 38, 39, 55, 56). We performed a detailed bioinformatic and statistical analysis of the mutational dispersion on both VDs and CDs, based on MOMP structure and on the mapping of all the B- and T-cell epitopes reported in the literature. We present statistically validated genomic evidence of the adaptation of this pathogen''s key antigen to the host pressure, which strongly indicates a strategy to evade the human immune system.     

Impact of metal pollution and Thlaspi caerulescens growth on soil microbial communities

Soil microorganisms drive critical functions in plant-soil systems. As such, various microbial properties have been proposed as indicators of soil functioning, making them potentially useful in evaluating the recovery of polluted soils via phytoremediation strategies. To evaluate microbial responses to metal phytoextraction using hyperaccumulators, a microcosm experiment was carried out to study the impacts of Zn and/or Cd pollution and Thlaspi caerulescens growth on key soil microbial properties: basal respiration; substrate-induced respiration (SIR); bacterial community structure as assessed by PCR-denaturing gradient gel electrophoresis (DGGE); community sizes of total bacteria, ammonia-oxidizing bacteria, and chitin-degrading bacteria as assessed by quantitative PCR (Q-PCR); and functional gene distributions as determined by functional gene arrays (GeoChip). T. caerulescens proved to be suitable for Zn and Cd phytoextraction: shoots accumulated up to 8,211 and 1,763 mg kg(-1) (dry weight [DW]) of Zn and Cd, respectively. In general, Zn pollution led to decreased levels of basal respiration and ammonia-oxidizing bacteria, while T. caerulescens growth increased the values of substrate-induced respiration (SIR) and total bacteria. In soils polluted with 1,000 mg Zn kg(-1) and 250 mg Cd kg(-1) (DW), soil bacterial community profiles and the distribution of microbial functional genes were most affected by the presence of metals. Metal-polluted and planted soils had the highest percentage of unique genes detected via the GeoChip (35%). It was possible to track microbial responses to planting with T. caerulescens and to gain insight into the effects of metal pollution on soilborne microbial communities.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.