Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS*

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.     

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16^INK4a, p21^WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.     

Chronic kidney disease-induced cardiac fibrosis is ameliorated by reducing circulating levels of a non-dialysable uremic toxin, indoxyl sulfate

Cardiovascular death commonly occurs in patients with chronic kidney disease. Indoxyl sulfate (IS), a uremic toxin, has been demonstrated in vitro as a contributory factor in cardiac fibrosis, a typical pathological finding in uremic cardiomyopathy. This study aimed to determine if cardiac fibrosis is reversible by lowering serum IS levels using an oral charcoal adsorbent, AST-120. Subtotal-nephrectomized (5/6-STNx) Sprague-Dawley rats were randomized to receive either AST-120 (AST-120, n=13) or no treatment (vehicle, n=17) for 12 weeks. Sham operated rats (n=12) were used as controls. Early left ventricular (LV) diastolic dysfunction was demonstrated by an increase in peak velocity of atrial filling [A and A' waves] and a decrease of E/A and E'/A' ratios obtained by echocardiography. This was accompanied by a 4.5-fold increase in serum IS (p<0.001) as well as elevated tail-cuff blood pressure (p<0.001) and heart weight (p<0.001). Increased LV fibrosis (p<0.001), gene expression of pro-fibrotic (TGF-β, CTGF) and hypertrophic (ANP, β-MHC and α-skeletal muscle actin) markers, as well as TGF-β and phosphorylated NF-κB protein expression were observed in STNx + vehicle rats. Treatment with AST-120 reduced serum creatinine (by 54%, p<0.05) and urine total protein (by 27%, p<0.05) vs vehicle whilst having no effect on blood pressure (AST-120=227 ± 11 vs vehicle =224 ± 8 mmHg, ns) and heart weight. The increase in serum IS was prevented with AST-120 (by 100%, p<0.001) which was accompanied by reduced LV fibrosis (68%, p<0.01) and TGF-β and phosphorylated NF-κB protein expression (back to sham levels, p<0.05) despite no significant change in LV function. In conclusion, STNx resulted in increased cardiac fibrosis and circulating IS levels. Reduction of IS with AST-120 normalizes cardiac fibrosis, in a blood pressure independent manner.     

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.     

Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20–27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD. Received: 13 July 1996 / Revised: 6 August 1996