Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense

Background

We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection.

Methods and results

Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5′ untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out.

General significance

Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.     

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids

Background

Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA.

Methods

In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5′ ends with ³²P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined.

Results and conclusions

Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg^2 +, Mn^2 + or Zn^2 + are absolutely necessary.

General significance

The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.     

Degradation of proteins upon storage at near-neutral pH: Indications of a proteolytic/gelatinolytic activity associated with aggregates

Background

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.

Methods

Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.

Results

Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.

Conclusions

Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.

General significance

The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.     

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

Background

Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes.

Methods

Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35 mg/kg, i.p.) (control + L and fructose + L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration.

Results

Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91^phox and p22^phox) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration.

Conclusions

Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration.

General significance

Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.     

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin

Background

Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown.

Methods

Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure–function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed.

Results

As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL.

Conclusions

The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL.

General significance

The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.     

The Roles of Individual Domains of RNase R in Substrate Binding and Exoribonuclease Activity: THE NUCLEASE DOMAIN IS SUFFICIENT FOR DIGESTION OF STRUCTURED RNA*

RNase R and RNase II are the two representatives from the RNR family of processive, 3′ to 5′ exoribonucleases in Escherichia coli. Although RNase II is specific for single-stranded RNA, RNase R readily degrades through structured RNA. Furthermore, RNase R appears to be the only known 3′ to 5′ exoribonuclease that is able to degrade through double-stranded RNA without the aid of a helicase activity. Consequently, its functional domains and mechanism of action are of great interest. Using a series of truncated RNase R proteins we show that the cold-shock and S1 domains contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, whereas the S1 domain is most likely required to position substrates for efficient catalysis. Most importantly, the nuclease domain alone, devoid of the cold-shock and S1 domains, is sufficient for RNase R to bind and degrade structured RNAs. Moreover, this is a unique property of the nuclease domain of RNase R because this domain in RNase II stalls as it approaches a duplex. We also show that the nuclease domain of RNase R binds RNA more tightly than the nuclease domain of RNase II. This tighter binding may help to explain the difference in catalytic properties between RNase R and RNase II.Ribonucleases (RNases) play important roles in RNA metabolism. They are responsible for the maturation of stable RNA and the degradation of RNA molecules that are defective or no longer required by the cell. Both maturation and degradation are initiated by endoribonucleolytic cleavage(s) and completed by the action of exoribonucleases (1). In Escherichia coli, three, relatively nonspecific, 3′ to 5′ processive exoribonucleases are responsible for degradation of RNA: RNase II, RNase R, and polynucleotide phosphorylase (PNPase).³ RNase II and PNPase appear to be primarily responsible for mRNA decay (2), although their precise functions may differ (3). However, mRNAs containing extensive secondary structure, such as repetitive extragenic palindromic sequences, are degraded by PNPase (4, 5) or RNase R (5). Likewise, degradation of highly structured regions of rRNA (6) and tRNA (7),⁴ is carried out by PNPase and/or RNase R. These findings suggest that PNPase and RNase R are the universal degraders of structured RNAs in vivo, leaving RNase II to act on relatively unstructured RNAs.Whether or not an RNase acts upon a particular RNA appears to depend upon the specificity of the RNase and the accessibility of the RNA to that RNase (1). Purified RNase R readily degrades both single- and double-stranded RNA molecules (5, 8), and it is the only known 3′ to 5′ exoribonuclease able to degrade through double-stranded RNA without the aid of helicase activity. To degrade RNA molecules containing double-stranded regions, RNase R requires a 3′ single-stranded overhang at least 5 nucleotides long to serve as a binding site from which degradation can be initiated (5, 8, 9).⁵ How RNase R then proceeds through the RNA duplex is of great interest. An important step toward elucidating the mechanism of action of RNase R is to determine the contribution that each of its domains makes to substrate binding and exoribonuclease activity.Despite differences in their physiological roles and intrinsic substrate specificities, RNase R and RNase II both belong to the widely distributed RNR family of exoribonucleases (10–12). RNR family members are all large multidomain proteins with processive 3′ to 5′ hydrolytic exoribonuclease activity that share a common linear domain organization. RNase R contains two cold-shock domains (CSD1 and CSD2) near its N terminus, a central nuclease, or RNB domain, an S1 domain near the C terminus, and a low complexity, highly basic region at the C terminus (Fig. 1A). The nuclease domain contains four highly conserved sequence motifs (10, 11). Motif I contains four conserved aspartate residues that are thought to coordinate two divalent metal ions that facilitate a two-metal ion mechanism similar to that of DEDD family exoribonucleases and the proofreading domains of many polymerases (13, 14). CSDs (15–17) and S1 domains (18, 19) are well known examples of RNA-binding domains. Interestingly, there are reports that both of these domains can act as nucleic acid chaperones and unwind RNA (20–29), providing a possible explanation for the ability of RNase R to degrade structured RNAs. The role of the basic region at the C terminus of RNase R is unknown, but it may act as an RNA-binding domain and/or a mediator of protein-protein interactions.Open in a separate windowFIGURE 1.Linear domain organization of RNase R and RNase II proteins. The CSDs are colored in cyan and blue for CSD1 and CSD2, respectively, the nuclease domains are in green, the S1 domains are red, and the low complexity, highly basic region, found in RNase R only, is in magenta. A, RNase R. RNase R full-length is the full-length wild-type RNase R protein. RNase RΔCSDs lacks both CSD1 and CSD2. RNase RΔBasic is missing the low complexity, highly basic region. RNase RΔS1 is missing both the S1 domain and the low complexity, highly basic region. RNase RΔCSDsΔS1 consists of the nuclease domain alone. B, RNase II. RNase II full-length is the full-length wild-type RNase II protein. RNase IIΔCSDsΔS1 contains the nuclease domain alone.Crystal structures of E. coli wild-type RNase II and a D209N catalytic site mutant in complex with single-stranded RNA have recently been solved (14, 30). In these structures the two CSDs and the S1 domain come together to form an RNA-binding clamp that directs RNA to the catalytic center at the base of a narrow, basic channel within the nuclease domain (14, 30). Only single-stranded RNA can be accommodated by the RNA-binding clamp and the nuclease domain channel, which explains the single strand specificity of RNase II. It is expected that RNase R will adopt a similar structure.In this study, we determine the contribution that each of the domains of RNase R makes to RNA-binding and exoribonuclease activity. We show that the CSDs and the S1 domain are important for substrate binding, although their roles differ. Of most interest, we show that the nuclease domain alone of RNase R is sufficient to degrade through double-stranded RNA, whereas the nuclease domain of RNase II is unable to carry out this reaction. The nuclease domain of RNase R also binds RNA more tightly, which may explain the difference in catalytic properties between RNase R and RNase II.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Tracking the elusive 5′ exonuclease activity of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> RNase J

Key message

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.

Abstract

RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Significance of MEFV gene R202Q polymorphism in Turkish familial Mediterranean fever patients

Objective

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and inflammation in the peritoneum, synovium, or pleura, accompanied by pain. The disease is associated with mutations in the Mediterranean fever (MEFV) gene, which encodes for the pyrin protein. The aim of this study was to explore the frequency and clinical significance of the R202Q (c.605G>A) polymorphism in exon 2 of the MEFV gene in a cohort of Turkish patients with FMF.

Methods

The study included 191 patients with FMF and 150 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay for the MEFV gene R202Qpolymorphism.

Results

The genotype and allele frequencies of R202Q polymorphism showed a statistically significant difference between FMF patients and controls (p < 0.0001 and p = 0.0004, respectively) and especially the homozygous AA genotype was significantly higher in FMF patients than healthy controls (p = 0.0002; odds ratio = 6.27; 95% CI = 2.1–18.3). However no significant association was observed between clinical and demographic features of FMF patients and R202Qpolymorphism.

Conclusion

The results of this study showed that there was a high association between MEFV gene R202Q polymorphism and FMF. R202Q polymorphism should be included in routine molecular diagnosis of FMF patients.     

Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.