Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect

We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2_pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2_pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1.Key words: VPS4, ESCRT, plant, Arabidopsis, SKD1, ATPase, MVB, proanthocyanidin, transparent testa, mucilage, tannin, seed coat, AtSKD1AAA ATPases are important regulators of a plethora of cellular functions such as peroxisome biogenesis, vesicle-mediated transport, control of cell divisions and gene expression. This variety is based on a common mechanism, the energy dependent unfolding, remodeling and disassembly of proteins and protein complexes.¹Mammalian SKD1 and its yeast homolog VPS4 are AAA ATPases involved in the sorting of monoubiquitylated trans-membrane cargo to the lysosome/vacuole by dismantling the members of the endosomal sorting complex required for transport (ESCRT) complexes from the endosomal membrane.² The Arabidopsis SKD1 homolog, AtSKD1, has been characterized recently and has been shown to be an ortholog of SKD1/Vps4.^3,4 Mutations for all three ATPases are known that alone or in combination render them dominant-negative.^3–6 Overexpression of dominant-negative AtSKD1 is, however, lethal for Arabidopsis plants. Therefore, we have used the trichome and non-root-hair-cell-specific GL2_pro promoter to address the function of AtSKD1 in planta. Cells that express the dominant-negative versions show multiple nuclei, fragmented vacuoles and ultimately die. These phenotypes are most likely due to a block in vacuolar trafficking of soluble cargo that is instead secreted.⁴GL2_pro is also active in the coat of developing Arabidopsis seeds. We therefore inspected seeds of lines overexpressing dominant-negative and wild-type AtSKD1.     

Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE³⁷³ neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.     

Deciphering a Molecular Mechanism of Neonatal Diabetes Mellitus by the Chemical Synthesis of a Protein Diastereomer, [d-AlaB8]Human Proinsulin

Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly^B8 is replaced by Ser^B8. Modular total chemical synthesis was used to prepare the wild-type [Gly^B8]proinsulin molecule and three analogs: [d-Ala^B8]proinsulin, [l-Ala^B8]proinsulin, and the clinical mutant [l-Ser^B8]proinsulin. The protein diastereomer [d-Ala^B8]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [l-Ser^B8]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [l-Ser^B8]proinsulin did not fold well under the physiological conditions investigated, once folded the [l-Ser^B8]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.     

Life without tRNAArg–adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA^Arg_ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA^Arg_CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA^Arg_CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA^Arg and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA^Arg_ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA^Arg_ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA^Arg_CCG and tadA, and then acquired a new tRNA^Arg_UCG. This permitted further tRNA^Arg_ACG mutations with tRNA^Arg_GCG or its disappearance, leaving a single tRNA^Arg_UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.     

Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG⁻ cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG⁻ cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG⁻ cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.     

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia

The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 10⁹ daltons for mu particle DNA and 0.81 × 10⁹ daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 10⁹ and 5.05 × 10⁹ daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.