Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.     

Promoter diversity in multigene transformation

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.     

Characterizing the complexity of enzymes on the basis of their mechanisms and structures with a bio-computational analysis

Enzymes are basically composed of 20 naturally occurring amino acids, yet they catalyse a dizzying array of chemical reactions, with regiospecificity and stereospecificity and under physiological conditions. In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors (both metal ions and organic molecules), to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving de novo design followed by exquisite regulation and modulation to create optimal fitness for life.     

CRISPR/Cas9 activity in the rice <Emphasis Type="Italic">OsBEIIb</Emphasis> gene does not induce off-target effects in the closely related paralog <Emphasis Type="Italic">OsBEIIa</Emphasis>

Genome editing with the CRISPR/Cas9 system allows mutations to be induced at any 20-bp target site in the genome preceded by the short protospacer adjacent motif (PAM) 5′-NGG-3′. The brevity and degeneracy of the PAM ensures that the motif occurs every ~10 bp in plant genomes, and all plant genes therefore contain many targetable sites. However, the CRISPR/Cas9 system tolerates up to three mismatches in the target site, so the ability to target genes in a specific manner requires the design of synthetic guide RNAs (sgRNAs) that do not bind off-target sites anywhere else in the genome. This is straightforward for single-copy genes but more challenging if a target gene has one or more paralogs because the principles that balance targeting efficiency (the frequency of on-target mutations) and accuracy (the absence of off-target mutations) are not fully understood and may be partially species-dependent. To investigate this phenomenon in rice, we targeted the rice starch branching enzyme IIb gene (OsBEIIb) with two sgRNAs designed to differ at two and six positions, respectively, from corresponding sites in the close paralog OsBEIIa. In each case, half of the mismatches were in the essential seed region immediately upstream of the PAM, where exact pairing is thought to be necessary, and the other half were in the distal part of the target. The sgRNAs also differed in predicted targeting efficiency (39 and 96 %, respectively). We found that the sgRNA with the low predicted efficiency was actually the most efficient in practice, achieving a mutation frequency of 5 % at the target site, whereas the sgRNA with the high predicted efficiency generated no mutations at the second target site. Furthermore, neither of the sgRNAs induced an off-target mutation in the OsBEIIa gene. Our data indicate that efficiency predictions should be tested empirically because they do not always reflect the experimental outcome and that a 1-bp mismatch in the seed region of a sgRNA is sufficient to avoid off-target effects even in closely related rice genes.     

The virome of maize rough dwarf disease: Molecular genome diversification,phylogeny and selection

The main threat to maize production in the Mediterranean area is maize rough dwarf disease (MRDD) caused by Maize rough dwarf virus (MRDV). The analysis of virus diversification is necessary to assess the risk of MRDD outbreaks. We analysed the virome of MRDD by next-generation sequencing using pooled samples prepared from maize plants collected in Spain between 1999 and 2017. This yielded the sequences of all 10 genomic segments from six MRDV isolates plus an additional variant of S5 in one sample. Five of the genomes were 29,145 nucleotides (nt) in length, and the other was 3 nt longer. The six genomes were AT rich with low codon usage bias and the 13 open reading frames (ORFs) showed low potential expression levels. The highest expression levels were predicted for ORFs encoding the non-structural proteins P6, P5-1 and P9-1, and the structural protein P10. There was a strong correlation between higher level expression and T-ended codons, which were the most over-represented class. The concatenate and three individual segments (S3, S6 and S7) each formed a monophyletic group, whereas the remaining segments split into two groups with different clustering relationships relative to two other European MRDV isolates. The MRDV virome showed certain temporal and geographical diversification, but several proteins were highly conserved, especially P7-2. Three significant recombination hotspots were identified among genomic segments and four within segments. The ORFs encoding structural proteins appear under greater purifying selection.     

Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site

The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.     

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.