No abstract

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNAindependent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNAdependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed. Nuclear factor 90 (NF90)1 is a cellular protein initially purified in complex with nuclear factor 45 (NF45) through its ability to bind to the antigen response recognition element in the interleukin-2 promoter (1). Independently, our laboratory identified NF90 as a cellular protein that interacts with VA RNA II , a structured adenoviral RNA, and with double-stranded RNA (dsRNA) (2). NF90 is one of the most abundant dsRNAbinding proteins in human cells, interacting preferentially with highly structured RNAs, in the order dsRNA Ͼ VA RNA II Ͼ VA RNA I Ͼ single stranded RNA (ssRNA). NF90 and NF45 have also been purified from human placental extracts in association with the protein synthesis eukaryotic initiation factor 2 and the catalytic subunit of the DNA-activated protein kinase DNA-PK (3). NF90 stabilizes the interactions between the catalytic subunit of DNA-PK and its regulatory heterodimeric DNA-binding subunits, Ku, in vitro, and it serves as a substrate for the activated DNA-PK in vitro.NF90 contains no recognized DNA binding motifs; rather it contains two dsRNA-binding motifs (dsRBMs) which lie downstream of a bipartite nuclear localization signal.2 Several homologues of NF90 have since been identified, including Xenopus 4F.1 (4), Spnr (5), and ILF3 (6) in mouse, and p74 in rat (7). All of these proteins share homology at their N terminus to a mouse protein, Zfr, a protein of unknown function which has a conserved homologue in Drosophila (8) (Fig. 1A). Downstream of this Zfr homology domain is a region of homology which these proteins share with NF45, which we term the NF45 homology domain.Several NF90 homologues and variants have been identified in human cells, and recent work from Duchange and colleagues (9) has helped clarify the relationships of several of these proteins. Alternatively spliced variants of the protein exist...

No abstract

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.