Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS^E proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.     

Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.     

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.     

Identification of the reactive cysteine residues in oligopeptidase B from Trypanosoma brucei

Oligopeptidase B (OpdB) from Trypanosoma brucei is a candidate therapeutic target in African trypanosomiasis. OpdB is an atypical serine peptidase, since activity is inhibited by thiol-blocking reagents and enhanced by reducing agents. We have identified C256 as the reactive cysteine residue that mediates OpdB inhibition by N-ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P(1) substrate-binding site, preventing substrate binding. We further demonstrate that C559 and C597 are responsible for the thiol-enhancement of OpdB activity. These studies may facilitate the development of specific OpdB inhibitors with therapeutic potential, by exploiting these unique properties of this enzyme.     

Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin

Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics.     

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.     

Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More mature fibrils (>24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.