Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases.

Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA-and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation, but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90-NF45 and NF110-NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Coregulation of NF90 and NF45 is a posttranscriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90-NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90-NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.Human nuclear factor 90 (NF90) and nuclear factor 45 (NF45) were originally purified as a sequence-specific DNA binding complex regulating the interleukin-2 (IL-2) promoter (10, 17). NF90 is the founder member of a family of proteins generated from differentially spliced transcripts of the ILF3 gene (12). NF90 and NF110, which differ at their C termini, are the two most prominent ILF3 isoforms in cells (12,33,42,55). Both have been repeatedly isolated in diverse studies and have been given a variety of names. For example, MPP4 (M-phase phosphoprotein 4) is similar, if not identical, to NF90 and is phosphorylated during M phase (23), and closely related proteins 4F.1 and 4F.2 were characterized in Xenopus as doublestranded RNA (dsRNA)-binding proteins (3). NF90 is also known as DRBP76, NFAR1, and TCP80 (34, 43, 55), and NF110 is also known as ILF3, NFAR2, TCP110, and CBTF 122 (4,43,53,55). Underlining the importance of these proteins, knockout of the mouse ILF3 gene led to muscle degeneration, respiratory failure, and death soon after birth (44).NF90 and NF110 contain two dsRNA binding motifs (dsRBMs) which are responsible for their ability to interact with structured RNA. They also have an RGG domain that is capable of nucleic acid binding, and NF110 has an additional GQSY region that can interact with nucleic acids. Although characterized as DNA-binding proteins (17,36,40,41), NF90 and NF45 do not contain a recognized sequence-specific DNAbinding domain and the complex containing NF90 and NF45 does not appear to interact with DNA directly. NF90 and NF45 have been purified in complexes containing the Ku proteins and DNA-protein kinase (PK), as well as eukaryotic initiation factor 2 (eIF2), and it is likely ...

Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein implicated in multiple cellular functions, but with few identified RNA partners. Using in vivo cross-linking followed by immunoprecipitation, we discovered a family of small NF90-associated RNAs (snaR). These highly structured non-coding RNAs of ∼117 nucleotides are expressed in immortalized human cell lines of diverse lineages. In human tissues, they are abundant in testis, with minor distribution in brain, placenta and some other organs. Two snaR subsets were isolated from human 293 cells, and additional species were found by bioinformatic analysis. Their genes often occur in multiple copies arranged in two inverted regions of tandem repeats on chromosome 19. snaR-A is transcribed by RNA polymerase III from an intragenic promoter, turns over rapidly, and shares sequence identity with Alu RNA and two potential piRNAs. It interacts with NF90's double-stranded RNA-binding motifs. snaR orthologs are present in chimpanzee but not other mammals, and include genes located in the promoter of two chorionic gonadotropin hormone genes. snaRs appear to have undergone accelerated evolution and differential expansion in the great apes.

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37°C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of saltwashed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.Bacterial proteins including diphtheria toxin (DT), 1 Pseudomonas exotoxin A (PE), Shiga toxin (ST), Shiga-like toxins (SLTs), and plant proteins such as ricin kill mammalian cells by catalytically inactivating key components of the translational machinery (1). DT and PE achieve this by the ADPribosylation of elongation factor-2 (2), whereas ST, SLTs, and ricin inhibit protein synthesis by removing a specific adenine residue from 28 S ribosomal RNA (3), leaving toxin-modified ribosomes unable to carry out protein synthesis. Since the target substrates for all these toxins are present in the cytosol, a key feature of toxicity is the delivery of a catalytically active polypeptide or fragment into this cellular location.Cellular entry by the protein toxins listed above involves the same generalized mechanism. The toxin binds to a normal cell surface component, which is thus utilized as a toxin receptor. Surface-bound toxin enters the cell by endocytosis in both clathrin-coated and uncoated pits/vesicles (2, 4). During subsequent intracellular transport, DT, PE, ST, and SLT are cleaved by the membrane-associated protease, furin, to separate the cell-binding domain from the catalytic domain (5). The furin cleavage site lies between two cysteines that are joined by a disulfide bond so that after proteolytic cleavage the resulting fragments remain covalently linked. The catalytically active (or A) chain (RTA) and the cell binding (or B) chain (RTB) of the plant toxin ricin are also synthesized as part of a single precursor protein (6), but the proteolytic cleavage necessary to separate them occurs during their biosynthesis in the producing plant (7). When the appropriate intracellular compartment is re...

Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction.