Brian E. Nordin

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA

Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.

Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNAIle and Thr-tRNAVal, respectively. Here we examined the chemical requirements at the 3′-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2′- and 3′-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2′-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3′-OH and not from the 2′-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2′-OH. Moreover, for IleRS the specificity for a 3′-O location of the scissile ester bond could be forced to the 2′-position by introduction of a 3′-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.

RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase.

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TΨC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system.

Global Human-Kinase Screening Identifies Therapeutic Host Targets against Influenza.

During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus–infected human cells.

Abstract 1789: Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform

HSP90 is a ubiquitous molecular chaperone that couples ATP hydrolysis to the folding and maturation of numerous client proteins. Because many of these client proteins are essential to cancer cell growth and survival, HSP90 inhibitors have undergone clinical investigation in numerous oncology indications. Here, using an LC-MS/MS platform previously developed to characterize ATP-competitive inhibitors of protein kinases, several HSP90 inhibitors were profiled in native cell lysates. This method utilizes an ATP derivative that covalently labels lysine residues in the ATP-binding site of any amenable enzyme. The specificity of inhibitors for all four paralogs of HSP90 (HSP90α, HSP90β, Grp94 and Trap-1) found in human cells was simultaneously monitored at their endogenous relative abundances. Interestingly, the concentration of HSP90 paralogs in human cell lysates was significantly higher than reported inhibitor binding constants, therefore complete inhibition was only observed at much higher concentrations than would have been predicted. In addition to the four HSP90 paralogs, inhibition data was collected on over 400 protein kinases and other nucleotide-binding proteins. This led to the discovery that NVP-AUY922 has off-target activity toward PMS2, an enzyme, like HSP90 itself, belonging to the GHKL ATPase superfamily. In addition to directly profiling compound targets in lysates, downstream effects of HSP90 inhibition were monitored by treating live cells and analyzing the resulting cell lysates at defined time points. Many proteins were found to be much less abundant in the compound-treated cells, including known HSP90 client proteins and novel, putative client proteins. Citation Format: Tyzoon K. Nomanbhoy, Brian E. Nordin, Jonathan Rosenblum, Yongsheng Liu. Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2014-1789