Inhibitor Mediated Protein Degradation

SummaryThe discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc3Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc3Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc3Arg. Degradation is rapid and robust, with 30%–80% of these abundant target proteins consumed within 1.3–5 hr. The proteasome is required for Boc3Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc3Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

MALT-1 shortens lifespan by inhibiting autophagy in the intestine of C. elegans

ABSTRACTThe caspase-like protease MALT1 promotes immune responses and oncogenesis in mammals by activating the transcription factor NF-κB. MALT1 is remarkably conserved from mammals to simple metazoans devoid of NF-κB homologs, like the nematode C. elegans. To discover more ancient, NF-κB -independent MALT1 functions, we analysed the phenotype of C. elegans upon silencing of MALT-1 expression systemically or in a tissue-specific manner. MALT-1 silencing in the intestine caused a significant increase in life span, whereas intestinal overexpression of MALT-1 shortened life expectancy. Interestingly, MALT-1-deficient animals showed higher constitutive levels of autophagy in the intestine, which were particularly evident in aged or starved nematodes. Silencing of the autophagy regulators ATG-13, BEC-1 or LGG-2, but not the TOR homolog LET-363, reversed lifespan extension caused by MALT-1 deficiency. These findings suggest that MALT-1 limits the lifespan of C. elegans by acting as an inhibitor of an early step of autophagy in the intestine.Abbreviations: AP-1: activator protein-1; AL: autolysosome; AP: autophagosome; ATG: Autophagy-related gene, BCL10: B cell lymphoma-10; CARD: caspase-recruitment domain; CARMA: CARD-MAGUK, C. elegans: Caenorhabditis elegans; CBM complex: CARMA-BCL10-MALT1 complex; EGFR: epidermal growth factor receptor; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; GPCRs: G-protein coupled receptors; Ig: Immunoglobulin; Int: intestinal; ITAMs: immunoreceptor tyrosine-mediated activation motifs; KO: knock-out; LC3: microtubule-associated protein 1A/1B-light chain 3; LUBAC: linear ubiquitin chain assembly complex; MALT-1: mucosa-associated lymphoid tissue protein-1; NF-κB: nuclear factor-kappa B; NFKI: NF-κB inhibitor; NGM: nematode growth medium; ns: not significant; NS: nervous system; OE: overexpressed; RER: rough endoplasmic reticulum; RNAi: RNA interference; TCR: T cell receptor; TEM: Transmission electron microscopy; TRAF6: TNF receptor-associated factor-6

Clofarabine Targets the Large Subunit (α) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers

Available in PMC 2013 July 27Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α6. Our data validate hRNR as an important target of ClF, provide evidence that in vivo α's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.Howard Hughes Medical Institute (Investigator)National Institutes of Health (U.S.) (NIH grant GM29595)National Institutes of Health (U.S.) (NIH grant GM67167)Damon Runyon Cancer Research Foundation (postdoctoral fellowship (DRG2015-09))Howard Hughes Medical Institute (International Student Fellowship

Boc₃Arg-Linked Ligands Induce Degradation by Localizing Target Proteins to the 20S Proteasome

Targeted protein degradation is a promising strategy for drug design and functional assessment. Several small molecule approaches have been developed that localize target proteins to ubiquitin ligases, inducing ubiquitination and subsequent degradation by the 26S proteasome. We discovered that the degradation of a target protein can also be induced by a recognition ligand linked to <i>tert</i>-butyl carbamate (Boc₃)-protected arginine (B₃A). Here, we show that this process requires the proteasome but does not involve ubiquitination of the target protein. B₃A does not perturb the structure of the target protein; instead, a B₃A-ligand stabilizes its target protein. B₃A ligands stimulate activity of purified 20S proteasome, demonstrating that the tag binds directly to the 20S proteasome. Moreover, purified 20S proteasome is sufficient to degrade target proteins in the presence of their respective B₃A-linked recognition ligands. These observations suggest a simple model for B₃A-mediated degradation wherein the B₃A tag localizes target proteins directly to the 20S proteasome. Thus, B₃A ligands are the first example of a ubiquitin-free strategy for targeted protein degradation

Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcificati