A novel nucleolar protein interacts with ribosomal protein S19

The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of patients with Diamond-Blackfan anemia (DBA), which is a rare congenital erythroblastopenia. DBA patients have a variety of clinical characteristics, and the role of the RPS19 gene in the pathogenesis of the disease is presently unknown. To investigate a possible role for RPS19 in erythropoiesis, we looked for proteins associated with mouse RPS19 using a yeast two-hybrid system and identified a novel protein, which we named S19 binding protein (S19BP). The deduced amino acid sequence of S19BP derived from cDNA defines a calculated mass of 15,849 and an isoelectric point of 11.3. No known functional motifs were found in S19BP except a short polylysine tract embedded in a putative nucleolar localization signal. Immunolocalization experiments revealed that S19BP was highly concentrated in nucleoli after 6 h of transfection in Cos-7 cells. S19BP was expressed ubiquitously at a basal level but a significantly high level of expression was observed in some tissues.     

Fibroblast growth factor-2

Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF-2 contains a number of basic residues (pI 9.6) and consists of 12 anti-parallel beta-sheets organized into a trigonal pyrimidal structure. FGF-2 binds to four cell surface receptors expressed as a number of splice variants. Many of the biological activities of FGF-2 have been found to depend on its receptor's intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. However, considerable evidence suggest that intracellular FGF-2 might have a direct biological role particularly within the nucleus. In addition, heparan sulfate proteoglycans have been demonstrated to enhance and inhibit FGF-2 activity. The possibility that FGF-2 activity can be manipulated through alterations in heparan sulfate-binding is currently being exploited in the development of clinical applications aimed at modulating either endogenous or administered FGF-2 activity.     

Fibroblast growth factor-2

Fibroblast growth factor-2 (FGF-2) is a heparin-binding growth factor which occurs in several isoforms resulting from alternative initiations of translation: an 18 kD cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kD). FGF-2 has pleiotropic roles in many cell types and tissues; it is a motogenic, angiogenic and survival factor which is involved in cell migration, cell differentiation and in a variety of developmental processes. Although devoid of signal peptide, it could be secreted. It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors, but also through still unknown intracrine process(es) on intracellular targets. FGF-2 has many biological functions which are probably isoform-specific. Nevertheless, FGF-2 is not essential for embryonic development as knock-out mice for the growth factor are viable and fertile although they exhibit abnormalities in neuronal differentiation. Use of FGF-2 as therapeutic agent for the treatment of ischemic cardiovascular disease is promising and clinical trials are in progress.     

Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.     

Fibroblast growth factor-2 stimulates phospholipase Cbeta in adult cardiomyocytes.

Although fibroblast growth factor-2 (FGF-2) plays an important role in cardioprotection and growth, little is known about the signals triggered by it in the adult heart. We therefore examined FGF-2-induced effects on phosphoinositide-specific phospholipase C (PI-PLC) isozymes, which produce second messengers linked to the inotropic and hypertrophic response of the myocardium. FGF-2, administered by retrograde perfusion to the isolated heart, induced an increase in inositol-1,4,5-trisphosphate levels in the cytosol, as well as an increase in total PI-PLC activity associated with sarcolemmal and cytosolic fractions. Furthermore FGF-2 induced a time-dependent elevation in cardiomyocyte membrane-associated PLC gamma1 and PLC beta1 activities, assayed in immunoprecipitated fractions, and moreover, increased the membrane levels of PLC beta1 and PLC beta3. Activation of PLC beta is suggestive of FGF-2-induced cross-talk between FGF-receptor tyrosine kinase and G-protein-coupled signaling in adult cardiomyocytes and underscores the importance of FGF-2 in cardiac physiology.     

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.     

A putative gene of tobacco chloroplast coding for ribosomal protein similar to E. coli ribosomal protein S19.

A partial sequence of a cloned 3.2 Md BamHI fragment from tobacco chloroplast DNA revealed the occurrence of a putative gene for ribosomal protein. The putative gene is located on the left margin of the large single-copy region in the chloroplast DNA. The coding region contains 276 bp (92 codons). The amino acid sequence deduced from the DNA sequence shows 55% homology with that of E. coli S19 (91 amino acid residues).     

The primary structure of rat ribosomal protein S19

The covalent structure of rat ribosomal protein S19 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S19 contains 144 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 15,944. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-18 copies of the S19 gene. The mRNA for the protein is about 640 nucleotides in length. Rat S19 is related to Saccharomyces cerevisiae S16A and to Halobacterium marismortui S12.     

Solution structure of the ribosomal protein S19 from Thermus thermophilus.

Ribosomal protein S19 is a 10.6 kDa protein in the small subunit of the prokaryotic ribosome. We have determined a high-resolution solution structure of S19 from Thermus thermophilus. Structures were calculated using 1160 distance and dihedral angle restraints derived from (1)H, (15)N and (13)C NMR spectra. The structures show that S19 is a mixed alpha/beta protein with long disordered tails. The folding topology is not homologous to that of any other known protein structure. Potential rRNA and protein binding sites have been identified on the S19 surface.     

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.     

Analysis of the interactome of ribosomal protein S19 mutants

Diamond–Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one‐fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild‐type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild‐type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 ( http://proteomecentral.proteomexchange.org/dataset/PXD000640 ).     

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.     

The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA(C) protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA(C) does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA(C) to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA(C). Assays of epitope-tagged wild-type and mutant variants of CgtA(C) indicate that the C terminus of CgtA(C) is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA(C) alleles to function in vivo. Depletion of CgtA(C) led to perturbations in the polysome profile, raising the possibility that CgtA(C) is involved in ribosome assembly or stability.     

Fibroblast growth factor-2 determines severity of joint disease in adjuvant-induced arthritis in rats.

Rheumatoid arthritis (RA), a systemic inflammatory disease of unknown etiology, mainly affects synovial joints. Although angiogenic growth factors, including fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), may play a critical role in the development and progression of RA joint disease, little information is now available regarding their exact role in initiation and/or progression of RA. In this study, we show that both polypeptides were up-regulated in the rat joint synovial tissue of an adjuvant-induced model of arthritis (AIA), as well as human subjects with RA. FGF-2 overexpression via Sendai virus-mediated gene transfer significantly worsened clinical symptoms and signs of rat AIA, including hind paw swelling and radiological bone destruction, as well as histological findings based on inflammatory reaction, synovial angiogenesis, pannus formation, and osteocartilaginous destruction, associated with up-regulation of endogenous VEGF. FGF-2 gene transfer to non-AIA joints was without effect. These findings suggested that FGF-2 modulated disease progression, but did not affect initiation. Reverse experiments using anti-FGF-2-neutralizing rabbit IgG attenuated clinical symptoms and histopathological abnormalities of AIA joints. To our knowledge, this is the first report indicating direct in vivo evidence of disease-modulatory effects of FGF-2 in AIA, as probably associated with endogenous VEGF function. FGF-2 may prove to be a possible therapeutic target to treat subjects with RA.     

Fibroblast growth factor-2 primes human mesenchymal stem cells for enhanced chondrogenesis

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.     

Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals.

Rheb is a recently described member of the Ras family that was originally identified as an immediate-early gene in brain but is also widely expressed in other tissues. Here we demonstrate that Rheb interacts with and appears to regulate Raf-1 kinase, an essential component of the H-Ras signaling pathway. In direct contrast to H-Ras, however, the interaction of Rheb with Raf-1 is potentiated by growth factors in combination with agents that increase cyclic AMP (cAMP) levels. Protein kinase A-dependent phosphorylation of serine 43 within the regulatory domain of Raf-1 reciprocally potentiates its interaction with Rheb and decreases its interaction with H-Ras. A single amino acid in the G2 effector domain is critical for the differential properties of Rheb. Since Rheb is an immediate-early gene, our studies suggest that Rheb functions in concert with H-Ras to dynamically integrate cAMP and growth factor signaling.     

Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66

Fibroblast growth factor 2 (FGF-2) has a dual role as a classical extracellular signaling protein and as an intracellular factor. Isoforms of FGF-2, resulting from alternatively used start codons on one mRNA species, locate differentially to nuclear compartments. In this study we aimed to analyze functions of intracellular FGF-2 by identification of interacting proteins. We identified the 66-kDa subunit of splicing factor 3a (SF3a66) as a binding partner in a yeast two-hybrid screen and confirmed this interaction by pull-down assays. The splicing factor interacted with the 18-kDa (FGF-2(18)) and with the 23-kDa (FGF-2(23)) isoforms, indicating an interaction with a domain common to both isoforms. Moreover, FGF-2 interacted with the C-terminus of SF3a66, a sequence that has not previously been assigned a functional role. In a functional neurite outgrowth assay, SF3a66 enhanced neurite lengths similar to FGF-2(18). We have previously identified the spliceosomal assembly factor survival of motoneuron (SMN) protein as a protein interacting specifically with the FGF-2(23) isoform [Claus et al., J. Biol. Chem. 278 (2003), 479-485]. The identification of two FGF-2 interacting proteins from the same biochemical pathway suggests a novel intranuclear role of FGF-2.