Full-length Protein Substrates Research Articles

Defining E3 ligase–substrate relationships through multiplex CRISPR screening

Specificity within the ubiquitin–proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates—and in particular the molecular features (degrons) that they recognize—remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin–proteasome system.

Effect of Ferulic Acid, a Phenolic Inducer of Fungal Laccase, on 26S Proteasome Activities In Vitro.

The 26S proteasome is an ATP-dependent protease complex (2.5 MDa) that degrades most cellular proteins in Eukaryotes, typically those modified by a polyubiquitin chain. The proteasome-mediated proteolysis regulates a variety of critical cellular processes such as transcriptional control, cell cycle, oncogenesis, apoptosis, protein quality control, and stress response. Previous studies conducted in our laboratory have shown that 26S proteasomes are involved in the regulation of ligninolytic enzymes (such as laccase) in white-rot fungi in response to nutrient starvation, cadmium exposure, and ER stress. Laccases are useful biocatalysts for a wide range of biotechnological applications. The goal of the current study was to determine the effect of ferulic acid (4-hydroxy-3-methoxycinnamic acid), a phenolic compound known to induce some ligninolytic enzymes, on proteasomes isolated from mycelia of the wood-decomposing basidiomycete Trametes versicolor. The peptidase activities of 26S proteasomes were assayed by measuring the hydrolysis of fluorogenic peptide substrates specific for each active site: Suc-LLVY-AMC, Z-GGR-AMC and Z-LLE-AMC for chymotrypsin-like, trypsin-like, and caspase-like site, respectively. Ferulic acid affected all peptidase activities of the 26S fungal proteasomes in a concentration-dependent manner. A possible inhibitory effect of ferulic acid on peptidase activities of the 26S human proteasomes was tested as well. Moreover, the ability of ferulic acid to inhibit (at concentrations known to induce laccase activity in white-rot fungi) the rate of 26S proteasome-catalyzed degradation of a model full-length protein substrate (β-casein) was demonstrated by a fluorescamine assay and by a gel-electrophoretic analysis. Our findings provide new insights into the role of ferulic acid in lignin-degrading fungi. However, the detailed molecular mechanisms involved remain to be elucidated by future studies.

HDAC8 substrate selectivity is determined by long- and short-range interactions leading to enhanced reactivity for full-length histone substrates compared with peptides

Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.

A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities.

BackgroundMethyltransferases (MTs) catalyze the S-adenosylmethionine (SAM)-dependent methylation of a wide variety of protein and DNA substrates. Methylation of lysine, arginine or cytosine regulates a variety of biological processes including transcriptional activation and gene silencing. Despite extensive studies of the cellular roles of MTs, their quantitative kinetic characterization remains challenging. In the past decade, several assays have been developed to monitor methyl transfer activity utilizing different approaches including radiolabeling, antibodies or mass-spectrometry analysis. However, each approach suffers from different limitation and no easy continuous assay for detection of MT activity exists.ResultsWe have developed a continuous coupled assay for the general detection of MTs activity. In this assay, the formation of S-adenosylhomocysteine (SAH) product is coupled NAD(P)H oxidation through three enzyme reactions including glutamate dehydrogenase leading to absorbance changes at 340 nm. The utility and versatility of this assay is demonstrated for SET7/9 and SETD6 with peptides and full length protein substrates and for M.HaeIII with a DNA substrate.ConclusionsThis study shows a simple and robust assay for the continuous monitoring of MT enzymatic activity. This assay can be used for the determination of steady-state kinetic enzymatic parameters (e.g., kcat and KM) for a wide variety of MTs and can be easily adapted for high-throughput detection of MT activity for various applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0048-y) contains supplementary material, which is available to authorized users.

Identification of Novel Proteasome Inhibitors from an Enaminone Library.

A library of structurally distinct enaminones was synthesized using sonication or Ru(II) catalysis to couple primary, secondary, and tertiary thioamides with α-halocarbonyls or α-diazocarbonyls. Screening the library for proteasome inhibition using a luciferase-based assay identified seven structurally diverse compounds. Two of these molecules targeted luciferase, while the remaining five exhibited varying potency and specificity for the trypsin-like, chymotrypsin-like, or caspase-like protease activities of the proteasome. Physiological relevance was confirmed by showing these molecules inhibited proteasomal degradation of the full-length protein substrate p21cip1 expressed in tissue culture cells. A cell viability analysis revealed that the proteasome inhibitors differentially affected cell survival. Results indicate a subset of enaminones and precursor molecules identified in this study are good candidates for further development into novel proteasome inhibitors with potential therapeutic value.

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ∼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP.

Identification of Exosite-Targeting Inhibitors of Anthrax Lethal Factor by High-Throughput Screening

Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

SRT1720, SRT2183, SRT1460, and Resveratrol Are Not Direct Activators of SIRT1

Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo

Purpose: We employed a grp170-secreting tumour cell system to determine whether tumour cells engineered to secrete grp170 generate an antitumour-specific immune response. Further, we examine the possibility that secreted grp170 can bind to and co-transport out of tumour cells full-length tumour antigens that may play a role in the anti-tumour immune response.Materials and methods: Wild type Colon-26 and Colon-26 engineered to secrete grp170 were subcutaneously inoculated into BALB/c mice. Tumour growth was monitored, and variations in immunoregulatory mechanisms were evaluated using immunohistochemistry, lymphocyte depletion, ELISpot assays, and Western blot analysis.Results: Immunisation of animals with grp170-secreting tumour cells results in rejection of the tumour by induction of antigen-specific, CD8-dependent immune responses. The secreted grp170 is able to deliver full-length tumour antigens to the tumour microenvironment, thus making them available for uptake by antigen presenting cells (APCs) to initiate tumour-specific immune responses.Conclusions: These data parallel our studies showing that hsp110 or grp170 are able to chaperone full-length proteins, and when complexed with protein antigens and used as vaccines, these complexes elicit immune responses in vivo against the protein antigens. This cell-based approach has the potential to be utilised as a tumour-specific vaccine in tumours of various histological origins.

ClpP Hydrolyzes a Protein Substrate Processively in the Absence of the ClpA ATPase: Mechanistic Studies of ATP-Independent Proteolysis

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.

Use of docking peptides to design modular substrates with high efficiency for mitogen-activated protein kinase extracellular signal-regulated kinase.

The mitogen-activated protein kinase extracellular regulated kinase (ERK) plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150-fold, while the specificity of the substrate for ERK was preserved. Simple modular peptide substrates, which can be easily tailored to possess high phosphorylation efficiencies, will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays.

Molecular mechanisms of the phospho‐dependent prolyl cis/trans isomerase Pin1

Since its discovery 10 years ago, Pin1, a prolyl cis/trans isomerase essential for cell cycle progression, has been implicated in a large number of molecular processes related to human diseases, including cancer and Alzheimer's disease. Pin1 is made up of a WW interaction domain and a C-terminal catalytic subunit, and several high-resolution structures are available that have helped define its function. The enzymatic activity of Pin1 towards short peptides containing the pSer/Thr-Pro motif has been well documented, and we discuss the available evidence for the molecular mechanisms of its isomerase activity. We further focus on those studies that examine its cis/trans isomerase function using full-length protein substrates. The interpretation of this research has been further complicated by the observation that many of its pSer/Thr-Pro substrate motifs are located in natively unstructured regions of polypeptides, and are characterized by minor populations of the cis conformer. Finally, we review the data on the possibility of alternative modes of substrate binding and the complex role that Pin1 plays in the degradation of its substrates. After considering the available work, it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression.

PKR and eIF2α: Integration of Kinase Dimerization, Activation, and Substrate Docking

The antiviral RNA-dependent protein kinase, PKR, binds to viral double-stranded RNA in the cell and halts protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. In this issue of Cell, two complementary papers Dar et al. (2005) and Dey et al. (2005) address the interaction between PKR and eIF2alpha. The structures of eIF2alpha bound to PKR reveal that PKR forms a dimer, the interface of which is essential for kinase activation, and demonstrate how this protein substrate docks to its kinase. The structures, coupled with mutagenesis analysis, also demonstrate how phosphorylation of the activation loop can allosterically couple two distal regions, the dimerization and substrate recognition interfaces.