N-truncated Protein Research Articles

Premature Termination Codon in 5' Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation.

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantation for RelB deficiency

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

60. Intramuscular and Systemic Induction of the N-Truncated Dystrophin By Out-Of-Frame Exon 2 Skipping Restores Muscle Function in the Dup2 Mouse, Providing Further Support for a Therapeutic Pathway for 5’ DMD Mutations

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon.We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform.We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1.U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Preliminary results supports that systemic delivery of AAV9.U7snRNA vector into Dup2 mice induce expression of this functional isoform into all muscle including heart and diaphragm, thereby improving muscle histopathology.Following treatment, a genome-wide normalized RPF-Seq data analysis (Ribosome Protected Fragment) was performed to check if the treatment restored the Haslett gene lists (gene altered in DMD) to a ‘non-dystrophic’ pattern. Our data clearly indicates that the treatment restored the global expression pattern to a more normal pattern. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD.

Clinical characteristics of loss-of-function mutations in ABCA7 in Belgian Alzheimer disease patients and families

Clinical characteristics of loss-of-function mutations in ABCA7 in Belgian Alzheimer disease patients and families

G.P.94 : Induction of the N-truncated dystrophin by out-of-frame exon 2 skipping restores muscle function in the Dup2 mouse, providing further support for a therapeutic pathway for 5′ DMD mutations

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1. U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Notably, utrophin levels remain unchanged. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD. The efficiency of this treatment in inducing expression of the N-truncated dystrophin in 6 patient cell lines with different 5′ mutations is underway, and will be presented as well. Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon. We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform. We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1. U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Notably, utrophin levels remain unchanged. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD. The efficiency of this treatment in inducing expression of the N-truncated dystrophin in 6 patient cell lines with different 5′ mutations is underway, and will be presented as well.

O.13 Using out-of-frame exon skipping to induce IRES-driven expression of an N-truncated dystrophin isoform for 5’ DMD mutations

Frame-truncating mutations within the first five exons of the DMD gene typically do not result in Duchenne muscular dystrophy, but instead result in milder dystrophinopathy syndromes. This was first demonstrated in individuals carrying the c.9G > A (p.Trp3X) mutation, a North American founder allele associated with clinical severity ranging from a very mild Becker muscular dystrophy phenotype to a complete absence of symptoms. We have previously shown that the mild phenotype associated with this and similar 5’ exon mutations is due to alternative initiation of translation from methionines encoded in exon 6, a process mediated by the presence of a muscle-specific and glucocorticoid-inducible internal ribosomal entry site (IRES) found within exon 5. The resultant N-truncated dystrophin protein lacks the first calponin homology domain of the canonical actin binding domain 1. Nevertheless, it is highly functional, raising the possibility of the therapeutic use of this isoform. We hypothesized that disruption of the mRNA open reading frame via exon skipping – resulting in a downstream premature termination codon – would stimulate IRES utilization. The feasibility of this approach is supported by the recent identification of an asymptomatic patient harbouring an exon 2 deletion, and we designed U7snRNA vectors targeting exon 2 for use in both patient-derived cell lines and in a new DMD mouse model harbouring an exon 2 duplication. Both in vitro and in vivo we can stimulate IRES activity, and by combining exon-skipping and glucocorticoid treatment we are able to restore expression of a properly localized yet N-truncated dystrophin. These results not only provide evidence for the functionality of the dystrophin IRES – and, hence, for the presence of a novel N-truncated dystrophin isoform under yet to be clarified physiologic conditions – but also suggest that out-of-frame skipping is a promising therapeutic approach for DMD patients harbouring 5’ mutations.

Towards an Understanding of the Function of the Phytochelatin Synthase of Schistosoma mansoni

Phytochelatin synthase (PCS) is a protease-like enzyme that catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. PCS proteins also function in xenobiotic metabolism by processing GSH S-conjugates. The aim of the present study is to elucidate the role of PCS in the parasitic worm Schistosoma mansoni. Recombinant S. mansoni PCS proteins expressed in bacteria could both synthesize phytochelatins and hydrolyze various GSH S-conjugates. We found that both the N-truncated protein and the N- and C-terminal truncated form of the enzyme (corresponding to only the catalytic domain) work through a thiol-dependant and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. PCS transcript abundance was increased by metals and xenobiotics in cultured adult worms. In addition, these treatments were found to increase transcript abundance of other enzymes involved in GSH metabolism. Highest levels of PCS transcripts were identified in the esophageal gland of adult worms. Taken together, these results suggest that S. mansoni PCS participates in both metal homoeostasis and xenobiotic metabolism rather than metal detoxification as previously suggested and that the enzyme may be part of a global stress response in the worm. Because humans do not have PCS, this enzyme is of particular interest as a drug target for schistosomiasis.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

A protein lacking an essential input to its tertiary structure does fold into a compact globule

It is shown by equilibrium ultracentrifugation, velocity sedimentation, and viscometry that an N-truncated structural protein Caf1 (Cafl13–149) of the Yersinia pestis capsular antigen fiber exists as a monomer in solution and is capable of folding from denatured state into a compact globular state by itself, without involvement of a chaperone or other subunits. This happens despite the fact that in the norm, important information on the tertiary structure of each Caf1 subunit (specifically, completion of its hydrophobic core) is provided by the “donor” segment Ala1-Thr12 of the neighboring fiber subunit.

A novel mutation in NFKBIA/IKBA results in a degradation-resistant N-truncated protein and is associated with ectodermal dysplasia with immunodeficiency

Alterations in nuclear factor kappa B (NF-kappaB) essential modulator (NEMO; HUGO-approved symbol IKBKG) underlie most cases of ectodermal dysplasia with immune deficiency (EDI), a human disorder characterized by anhidrosis with diminished immunity. EDI has also been associated with a single heterozygous mutation at position Ser32 of the NF-kappaB inhibitor IkappaBalpha, one of two phosphorylation sites that are essential for targeting IkappaBalpha for proteasomal degradation and hence for activation of NF-kappaB. We report a novel heterozygous nonsense mutation in the IKBA (HUGO-approved symbol, NFKBIA) gene of a 1-year-old male child with EDI that introduces a premature termination codon at position Glu14. An in-frame methionine downstream of the nonsense mutation allows for reinitiation of translation. The resulting N-terminally truncated protein lacks both serine phosphorylation sites and inhibits NF-kappaB signaling by functioning as a dominant negative on NF-kappaB activity in lymphocytes and monocytes. These findings support the scanning model for translation initiation in eukaryotes and confirm the critical role of the NF-kappaB in the human immune response.

Both KH and non-KH domain sequences are required for polyribosome association of Scp160p in yeast.

Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160DeltaN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160DeltaC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studies further demonstrate that although the N-truncated protein remains competent to form RNPs, the C-truncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome association as an essential component of Scp160p function in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.

Transcription starting from an alternative promoter leads to the expression of the human ABO histo-blood group antigen.

Using the 5'-rapid amplification of cDNA ends technique with the ex vivo culture of AC133-CD34+ cells, a transcription start site was recently identified approximately 0.7 kb upstream from the transcription start sites previously determined. The transcripts from the alternative starting exon 1a were demonstrated in the cells of both erythroid and epithelial lineages. Because the nucleotide sequence of exon 1a does not contain an ATG codon, we examined whether transcription starting from exon 1a leads to production of a functional glycosyltransferase. Stable transfection experiments into the human gastric cancer MKN28 cells were performed using the various A transferase expression plasmids. Large amounts of A antigens were demonstrated on the cells transfected with the A transferase expression plasmid containing the entire cDNA from exon 1a or the 5'-truncated cDNA leading to the production of the N-truncated protein with deletion of the cytoplasmic tail and a portion of the transmembrane domain. However, negligible amounts of A antigens were observed on the cells transfected with the A transferase expression plasmids containing the 5'-truncated cDNA leading to the production of the N-truncated proteins without the cytoplasmic tail and the transmembrane domain. This study suggests that a functional A transferase could be produced by the transcription from exon 1a.

Characterization of a new nepovirus infecting apricot in Southeastern France: apricot latent ringspot virus

A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 � end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 � non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed. The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875.

In- trans regulation of the N-truncated-NIFA protein of Herbaspirillum seropedicae by the N-terminal domain

The NifA protein is responsible for transcription activation of nif genes in the endophytic diazotroph Herbaspirillum seropedicae. When expressed in Escherichia coli this NifA protein is unable to activate the transcription of a Klebsiella pneumoniae nifH::lacZ fusion. However, a form of NifA lacking the N-terminal domain did activate transcription and its activity was not inhibited by ammonium. In this work we show that when expressed separately, the N-terminal domain of H. seropedicae NifA protein can restore ammonium control of the N-truncated NifA activity in E. coli. This effect is dependent on the relative concentrations of the N-terminal domain and the N-truncated protein and suggests that the N-terminal domain behaves in this respect in a manner similar to that of NifL of the γ proteobacteria.

In-trans regulation of the N-truncated-NIFA protein of Herbaspirillum seropedicae by the N-terminal domain.

The NifA protein is responsible for transcription activation of nif genes in the endophytic diazotroph Herbaspirillum seropedicae. When expressed in Escherichia coli this NifA protein is unable to activate the transcription of a Klebsiella pneumoniae nifH::lacZ fusion. However, a form of NifA lacking the N-terminal domain did activate transcription and its activity was not inhibited by ammonium. In this work we show that when expressed separately, the N-terminal domain of H. seropedicae NifA protein can restore ammonium control of the N-truncated NifA activity in E. coli. This effect is dependent on the relative concentrations of the N-terminal domain and the N-truncated protein and suggests that the N-terminal domain behaves in this respect in a manner similar to that of NifL of the gamma proteobacteria.

Deletions at the N terminus of bacteriophage φ29 protein p6: DNA binding and activity in φ29 DNA replication

Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage φ29 protein p6. The activity of such proteins in the in vitro φ29 DNA replication system, their capacity to interact with the φ29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied. The initiation activity of protein p6 decreased considerably when 5 aa were deleted and was undetectable when 13 aa were removed. The mutant proteins were unable to specifically interact with the φ29 DNA ends. These results indicate the need of an intact N terminus for the activity of protein p6. However, such N-truncated protein inhibited both the specific binding of the wt protein p6 to the φ29 DNA ends and its activity in φ29 DNA replication.