Delivering proteins for export from the cytosol.

Correct protein function depends on delivery to the appropriate cellular or subcellular compartment. Following the initiation of protein synthesis in the cytosol, many bacterial and eukaryotic proteins must be integrated into or transported across a membrane to reach their site of function. Whereas in the post-translational delivery pathway ATP-dependent factors bind to completed polypeptides and chaperone them until membrane translocation is initiated, a GTP-dependent co-translational pathway operates to couple ongoing protein synthesis to membrane transport. These distinct pathways provide different solutions for the maintenance of proteins in a state that is competent for membrane translocation and their delivery for export from the cytosol.

Positional editing of transmembrane domains during ion channel assembly.

The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4µ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Establishing a flow process to coumarin-8-carbaldehydes as important synthetic scaffolds.

Despite their usefulness as fluorophores and synthetic precursors, efficient and reliable routes to coumarin-8-carbaldehydes are lacking. We describe here a high-yielding continuous flow synthesis that requires no manual intermediate purification or work-up, giving access to multigram quantities of the aldehyde product.

Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum.

Production and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-associated degradation (ERAD) eeyarestatin 1 (ES(I)) is a potent inhibitor of protein translocation. We have characterised this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ES(I) targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ES(I) acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ES(I), and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.

Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors.

Discovery of small-molecule degraders that activate ubiquitin ligase­mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell­based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007's binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007's binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor­derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell­based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.

Endoplasmic reticulum-associated degradation of a degron-containing polytopic membrane protein.

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.

CRISPR: A Screener's Guide.

The discovery of CRISPR-Cas9 systems has fueled a rapid expansion of gene editing adoption and has impacted pharmaceutical and biotechnology research substantially. Here, gene editing is used at an industrial scale to identify and validate new biological targets for precision medicines, with functional genomic screening having an increasingly important role. Functional genomic strategies provide a crucial link between observed biological phenomena and the genes that influence and drive those phenomena. Although such studies are not new, the use of CRISPR-Cas9 systems in this arena is providing more robust datasets for target identification and validation. CRISPR-based screening approaches are also useful later in the drug development pipeline for understanding drug resistance and sensitivity ahead of entering clinical trials. This review examines the developing landscape for CRISPR screening technologies within the pharmaceutical industry and explores the next steps for this constantly evolving screening platform.

A Flexible, Pooled CRISPR Library for Drug Development Screens.

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.

Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis.

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4-7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

Increasing the performance of pooled CRISPR-Cas9 drop-out screening.

Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.

Selective inhibition of the unfolded protein response: targeting catalytic sites for Schiff base modification.

Constitutive protein misfolding in the endoplasmic reticulum (ER) can lead to cellular toxicity and disease. Consequently, the protein folding environment within the ER is highly optimised and tightly regulated by the unfolded protein response (UPR). The apparent convergence of myriad diseases upon proteostasis in the ER has triggered a broad effort to identify selective inhibitors of the UPR. In particular, the most ancient component of this cellular stress pathway, the transmembrane protein IRE1, represents an appealing target for pharmacological intervention. Several inhibitors of IRE1 have recently been reported, each containing an aldehyde moiety that forms an unusual, highly selective Schiff base with a single key lysine (K907) within the RNase domain. Here we review the progress made in chemical genetic manipulation of IRE1 and the unfolded protein response and discuss computational strategies to rationalise the selectivity of covalently active small molecules for their targets. As an exemplar, we provide additional evidence that K907 of IRE1 is buried within a particularly unusual environment that facilitates Schiff base formation. New free-energy calculations within a molecular dynamics (MD) simulation framework show that the pKa of K907 is reduced by ~3.6 pKa units, relative to the model pKa of lysine in water. This significant pKa perturbation provides additional insights into the precise requirements for inhibition and for RNase catalysis by IRE1. Our computational method may represent a general approach for identifying potential covalent inhibitory lysine sites within buried protein cavities.

Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox.

Interfering with disulfide bond formation impedes protein folding and promotes endoplasmic reticulum (ER) stress. Due to limitations in measurement techniques, the relationships of altered thiol redox and ER stress have been difficult to assess. We report that fluorescent lifetime measurements circumvented the crippling dimness of an ER-tuned fluorescent redox-responsive probe (roGFPiE), faithfully tracking the activity of the major ER-localized protein disulfide isomerase, PDI. In vivo lifetime imaging by time-correlated single-photon counting (TCSPC) recorded subtle changes in ER redox poise induced by exposure of mammalian cells to a reducing environment but revealed an unanticipated stability of redox to fluctuations in unfolded protein load. By contrast, TCSPC of roGFPiE uncovered a hitherto unsuspected reductive shift in the mammalian ER upon loss of luminal calcium, whether induced by pharmacological inhibition of calcium reuptake into the ER or by physiological activation of release channels. These findings recommend fluorescent lifetime imaging as a sensitive method to track ER redox homeostasis in mammalian cells.

The unfolded protein response element IRE1α senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling.

The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cytosolic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.

New twists in the unfolded protein response.

The response of S. pombe, also known as fission yeast, to misfolded proteins involves mechanisms that have not been observed in other species.

Dissecting the physiological role of selective transmembrane-segment retention at the ER translocon.

The membrane integration of polytopic proteins is coordinated at the endoplasmic reticulum (ER) by the conserved Sec61 translocon, which facilitates the lateral release of transmembrane (TM) segments into the lipid phase during polypeptide translocation. Here we use a site-specific crosslinking strategy to study the membrane integration of a new model protein and show that the TM segments of the P2X2 receptor are retained at the Sec61 complex for the entire duration of the biosynthetic process. This extremely prolonged association implicates the Sec61 complex in the regulation of the membrane integration process, and we use both in vitro and in vivo analyses to study this effect further. TM-segment retention depends on the association of the ribosome with the Sec61 complex, and complete lateral exit of the P2X2 TM segments was only induced by the artificial termination of translation. In the event of the premature release of P2X2 TM1 from the ER translocon, the truncated polypeptide fragment was to found aggregate in the ER membrane, suggesting a distinct physiological requirement for the delayed release of TM segments from the ER translocon site.

The oligomeric state of Derlin-1 is modulated by endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.