Degron Sequence Research Articles

Dual ON/OFF-switch chimeric antigen receptor controlled by two clinically approved drugs

The ability to remotely control the activity of chimeric antigen receptors (CARs) with small molecules can improve the safety and efficacy of gene-modified T cells. Split ON- or OFF-switch CARs involve the dissociation of tumor-antigen binding from T cell activation (i.e., CD3ζ) on the receptor (R-) and signaling (S-) chains, respectively, that either associate or are disrupted in the presence of a small molecule. Here, we have developed an inducible (i)ON-CAR comprising the anti-apoptotic B cell lymphoma protein 2 protein in the ectodomain of both chains which associate in the presence of venetoclax. We showed that inducible ON (iON)-CAR T cells respond to target tumors cells in the presence of venetoclax or the BH3 mimetic navitoclax in a dose-dependent manner, while there is no impact of the drugs on equivalent second generation-CAR T cells. Within 48 h of venetoclax withdrawal, iON-CAR T cells lose the ability to respond to target tumor cells in vitro as evaluated by Interferon-gamma (IFNγ) production, and they are reliant upon the presence of venetoclax for in vivo activity. Finally, by fusing a degron sequence to the endodomain of the iON-CAR S-chain we generated an all-in-one ON/OFF-switch CAR, the iONØ-CAR, down-regulated by lenalidomide within 4 to 6 for functionally inactive T cells (no IFNγ production) within 24 h. We propose that our remote-control CAR designs can reduce toxicity in the clinic. Moreover, the periodic rest of iON and iONØ-CAR T cells may alleviate exhaustion and hence augment persistence and long-term tumor control in patients.

A novel mutation in SoIAA20 confers cross-resistance to 2,4-Dichlorophenoxyacetic acid and other auxinic herbicides in Sonchus oleraceus.

2,4-Dichlorophenoxyacetic acid (2,4-D) and other auxinic herbicides are important for weed control in cropping systems globally. Weeds with resistance to 2,4-D and other auxinic herbicides have evolved, including several populations of Sonchus oleraceus from multiple sites in Australia. We report the underlying mechanism in these populations that gives rise to auxinic herbicide resistance. We studied a total of three susceptible and eight resistant Sonchus oleraceus populations. All resistant populations had a deletion of three amino acids flanking the degron sequence of an Aux/IAA gene, SoIAA20, which was not found in the three susceptible populations. The eight populations with the resistant allele were also resistant to dicamba, fluroxypyr and clopyralid. The resistant plants also had reduced movement of 2,4-D out of the treated tissues compared to susceptible plants. The paired deletion flanking the degron region of SoIAA20 likely provides resistance to 2,4-D by restricting the movement of 2,4-D from the treated tissue to the rest of the plant. We hypothesise that this deletion keeps the 2,4-D bound to the target site. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A C-Degron Structure-Based Approach for the Development of Ligands Targeting the E3 Ligase TRIM7.

TRIM7 is a ubiquitin E3 ligase with key regulatory functions, mediating viral infection, tumor biology, innate immunity, and cellular processes, such as autophagy and ferroptosis. It contains a PRYSPRY domain that specifically recognizes degron sequences containing C-terminal glutamine. Ligands that bind to the TRIM7 PRYSPRY domain may have applications in the treatment of viral infections, as modulators of inflammation, and in the design of a new class of PROTACs (PROteolysis TArgeting Chimeras) that mediate the selective degradation of therapeutically relevant proteins (POIs). Here, we developed an assay toolbox for the comprehensive evaluation of TRIM7 ligands. Using TRIM7 degron sequences together with a structure-based design, we developed the first series of peptidomimetic ligands with low micromolar affinity. The terminal carboxylate moiety was required for ligand activity but prevented cell penetration. A prodrug strategy using an ethyl ester resulted in enhanced permeability, which was evaluated using confocal imaging.

An intrinsic network of polar interactions is responsible for binding of UL49.5 C-degron by the CRL2KLHDC3 ubiquitin ligase.

Bovine herpesvirus type 1 (BoHV-1) is a pathogen of cattle responsible for infectious bovine rhinotracheitis. The BoHV-1 UL49.5 is a transmembrane protein that binds to the transporter associated with antigen processing (TAP) and downregulates cell surface expression of the antigenic peptide complexes with the major histocompatibility complex class I (MHC-I). KLHDC3 is a kelch domain-containing protein 3 and a substrate receptor of a cullin2-RING (CRL2) E3 ubiquitin ligase. Recently, it has been identified that CRL2KLHDC3 is responsible for UL49.5-triggered TAP degradation via a C-degron pathway and the presence of the degron sequence does not lead to the degradation of UL49.5 itself. The molecular modeling of KLHDC3 in complexes with four UL49.5 C-terminal decapeptides (one native protein and three mutants) revealed their activity to be closely correlated with the conformation which they adopt in KLHDC3 binding cleft. To analyze the interaction between UL49.5 and KLHDC3 in detail, in this work a total of 3.6 μs long molecular dynamics simulations have been performed. The complete UL49.5-KLHDC3 complexes were embedded into the fully hydrated all-atom lipid membrane model with explicit water molecules. The network of polar interactions has been proposed to be responsible for the recognition and binding of the degron in KLHDC3. The interaction network within the binding pocket appeared to be very similar between two CRL2 substrate receptors: KLHDC3 and KLHDC2.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10

In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ("Ala-tails") that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.

Heterofunctional polymeric degrading chimeras (HYDRAC) for targeted protein degradation.

e15106 Background: Here we utilize a novel proteomimetic polymer platform technology which packages peptide side chains as globular structures with benificial emergent properties for the development of compounds capable of targeted protein degradation. We refer to this class of compounds as Heterofunctional polYmeric DegRAding Chimeras (HYDRACs) and demonstrate the selective degradation of the oncoprotein Myc as a proof-of-concept. Methods: HYDRACs were generated using ring-opening metathesis polymerization (ROMP), resulting in structures highly resistant to proteolysis. Heterofunctional constructs were designed incorporating secondary sequences in addition to Myc-targeting H1. The degron sequence (RRRG) was incorporated to engage endogenous cellular machinery for targeted protein degradation. Immunofluorescence/confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with use of both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1 sequence, homopolymers, monomeric peptides) controls were used to assess tumor antiproliferative effects and Myc-specific responses. Myc target engagement was assessed via immunoprecipitation assays, NMR, circular dichroism, and fluorescence polarization with protein degradation monitored by Western blot. A CRISPR knockdown library screen of all known E3 ligases and mass spec was used to identify pathways responsible for HYDRAC-induced protein degradation. Results: A library of HYDRACs with narrow polydispersity and predetermined degrees of polymerization were generated. These compounds are cell-penetrating with antiproliferative effects at micromolar IC50s. HYDRAC treatment resulted in significant decreases in Myc protein levels, which was rescued by inhibition with MG132 and MLN4924. RNAseq showed selective overexpression of Myc pathway genes. Biophysical analysis showed strong HYDRAC-Myc interactions, supported by the presence of Myc protein following pull-down of biotin-terminated HYDRACs. Identification of E3 ligases implicated in the degradation pathway using a combination of a CRISPR knock down library and mass spectrometry are ongoing. Mice bearing Myc-CaP tumors showed delayed tumor growth following treatment with Myc-degrading HYDRACs. Studies on biodistribution and pharmacokinetic profile of the compound, as well as the ability to degrade other proteins of interest using combinations of degron sequences are ongoing. Conclusions: The presented work demonstrates the feasibility of HYDRACs for targeted protein degradation of Myc, providing rationale for further studies in Myc-driven tumor models. This successful demonstration of this first-in-class compound also opens the door for the development of HYDRACs targeting other proteins of interest and incorporation of different E3 ligase recruiters for tissue or disease-specific activity.

Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron.

Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Abstract 2714: Inhibiting Myc using heterofunctional polymeric degrading chimeras (HYDRACs): a novel class of compounds capable of targeted protein degradation

Abstract Background: The proto-oncogene Myc is known to play critical roles in tumorigenesis, being dysregulated in >70% of human cancers. Development of small molecule Myc drugs has been hampered due to the lack of suitable binding pockets. Myc inhibitory peptides H1, derived from the bHLH-LZ region, have been explored to address this. However, poor pharmacokinetic profiles have precluded clinical translation. Here, we use a novel modular platform technology referred to as the HYDRAC for the development of dual-functional Myc inhibitors/degraders. Methods: HYDRACs were generated using ROMP resulting in dense brush polymers where peptides occupy every side chain. Heterofunctional constructs were designed incorporating synergistic secondary sequences in addition to Myc-targeting H1. Specifically, a RRRG degron sequence was added to engage endogenous cellular machinery for targeted Myc degradation. Immunofluorescence and confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1, monomeric peptides, etc) controls were used to assess anti-proliferative and Myc-specific responses. Myc engagement was assessed via biophysical assays and protein degradation monitored by Western blot. A CRISPR E3 ligase knockdown library and mass spec was used to identify pathways responsible for HYDRAC-induced protein degradation. Results: A library of HYDRACs with low PDI and predetermined DPs were generated. These compounds were shown to be cell-penetrating with antiproliferative effects at sub-micromolar IC50 values in a formulation- and Myc-dependent manner. HYDRAC treatment resulted in significant decreases in Myc protein levels, rescued by proteasomal inhibition with MG132. RNAseq showed selective overexpression of Myc pathway genes. Biophysical analysis showed strong HYDRAC-Myc interactions, in addtion to Myc protein present following pull-down of biotin-terminated HYDRACs. Selectivity studies revealed that HYDRAC activity requires an intact H1 targeting sequence. Identification of E3 ligases implicated in the degradation pathway are ongoing. Mice bearing transplanted Myc-CaP tumors showed delayed tumor growth following IP administration of Myc-degrading HYDRACs, with studies exploring the biodistribution and pharmacokinetic profile of the compound ongoing. Conclusion: We present a novel platform technology that addresses the challenges inherent to peptide delivery approaches. The presented work demonstrates the feasibility of HYDRACs for targeted Myc inhibition and degradation. This first-in-class compound also opens the door for the development of HYDRACs targeting other proteins of interest and multiplexing different E3 ligase recruiters for tissue or disease-specific activity. Citation Format: Max M. Wang, Mihai I. Truica, Brayley Gattis, Xiaoyu Zhang, Sarki Abdulkadir, Nathan C. Gianneschi. Inhibiting Myc using heterofunctional polymeric degrading chimeras (HYDRACs): a novel class of compounds capable of targeted protein degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2714.

Phosphorylated Serine Marks Proteins for Degradation By the Mitochondrial Matrix Protease, Clpxp

Mitochondrial ClpXP is an ATP-dependent protease complex in which the ClpX AAA+ chaperone binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. The serine protease ClpXP is located in the mitochondrial matrix and maintains the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is overexpressed in 45% of AML patients. Genetic or chemical inhibition or hyperactivation of ClpXP kills leukemic cells and stem cells in vitro and in vivo. Yet, the degron sequences or post-translational modifications that are targeted by mitochondrial ClpXP remain to be elucidated. In Bacillus subtilis, the bacterial ClpXP homologue degrades proteins containing phospho-arginine. To determine if phosphorylated amino acids also mark proteins for mitochondrial ClpXP degradation, we evaluated human ClpXP protease activity in the presence of phosphorylated amino acids. Recombinant human ClpXP was incubated with FITC-tagged casein substrates and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). The release of fluorogenic FITC-tag was measured to assess ClpXP protease activity. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP. Similar inhibition was seen with short peptides containing pSer and pThr. In contrast, pTyr, pArg, free phosphates, and the dephosphorylated amino acids or peptides did not affect ClpXP protease activity in this assay. Although pSer and pThr inhibited the protease activity of ClpXP, they had no impact on ClpX ATPase activity nor the peptidase activity of isolated ClpP. We characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green Phosphate assay, respectively. pSer and pThr had no effect on ClpP peptidase activity nor ClpX ATPase activity. Therefore, pSer and pThr specifically inhibited ClpXP's ability to degrade full-length proteins. Next, we investigated if pSer and pThr could bind to ClpX or ClpP. Thermal shift binding assays indicated that pSer and pThr but not pTyr and pArg bound to ClpX and none of the phosphorylated amino acids bound to ClpP. Similarly, Ala-pSer-Ala (ApSA), Arg-Arg-Ala-pSer-Val-Ala (RRApSVA) and Ala-pThr-Ala (ApTA) peptides bound to ClpX, while the non-phosphorylated peptides did not. Thus, taken together, pSer and pThr interact with ClpX at a site outside its ATP binding pocket. Previously we showed that ClpX and ClpP interact with respiratory chain complex II subunit SDHA. Moreover, inhibition of ClpP in AML cells increased misfolded or damaged SDHA protein and impaired respiratory chain complex II activity. Therefore, we asked if inhibition of ClpP and ClpX impacted levels of SDHA serine phosphorylation. We knocked down ClpX and ClpP individually in OCI-AML2 cells using shRNA. Then, mitochondrial lysates were prepared, serine phosphorylated proteins were immunoprecipitated and probed for SDHA. Increased abundance of serine phosphorylated SDHA was observed in AML cells after ClpP and ClpX knockdown. We also demonstrated that addition of recombinant ClpXP to the immunoprecipitates selectively degraded the fraction of serine phosphorylated SDHA. We then showed that serine phosphorylated SDHA was present only in the digitonin insoluble fraction of mitochondria proteins, indicating it is only found on damaged protein aggregates. In summary, we discovered that pSer and pThr promote the degradation of damaged respiratory chain proteins by mitochondrial ClpXP. Thus, this work highlights new mitochondrial biology and identifies a potential new strategy to develop ClpXP inhibitors for the treatment of AML.

The mIAA7 degron improves auxin-mediated degradation in Caenorhabditiselegans.

Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Development of heterobifunctional proteomimetic polymers for delivery of MYC inhibitory peptides and targeted MYC degradation.

e15049 Background: The c-Myc(Myc) oncogene plays critical roles in tumorigenesis and therapeutic resistance, being dysregulated in up to 70% of all human cancers. Heterodimerization with the closely related bHLH-LZ protein Max is required for oncogenic transformation, yet the development of small molecule inhibitors has been challenging since disordered Myc and Max monomers lack suitable binding pockets, while the Myc-Max interface is flat and extensive. Myc inhibitory peptides derived from the first helix (H1) of the bHLH-LZ region have overcome the limited binding affinity of small molecules. However, poor cellular penetrance and extremely limited bioavailability have precluded clinical translation of peptide-based approaches. Aim: To address and improve the limitations and low potential for clinical translation of Myc inhibitory peptides via a versatile peptide delivery platform. This novel platform technology is referred to as the Protein-Like Polymer (PLP) and it is based on packaging peptides together as high-density brush polymers. Methods: We generated H1-PLPs using ring-opening metathesis polymerization and characterized them using HPLC, LC-MS, NMR, and GPC. We also explored the potential of PLPs to be designed as heterobifunctional compounds by copolymerizing with secondary side chain peptides (nuclear localization sequences and degron sequence). For biological studies, immunofluorescence and confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with use of both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1 sequence, homopolymers, monomeric peptides) controls were used to assess tumor antiproliferative effects and Myc-based selectivity of response. Myc target engagement was assessed via immunoprecipitation assays and dual-reporter luciferase-based assays. Results: Our studies show that heterobifunctional PLPs employing H1 peptide and a degron sequence are cell-penetrating and co-localize with Myc in the nucleus. They display antiproliferative effects with submicromolar IC50 values in a Myc-dependent manner. The PLPs lead to Myc/Max complex disruption, promote significant levels of Myc degradation and prevent binding of Myc/Max heterodimers to the Myc/Max consensus canonical DNA binding site. Selectivity studies reveal that antiproliferative effects are dependent on intact H1 sequence. Animal studies using transplanted Myc-CaP model, which mimics the clinical course of human castration resistant prostate cancer are ongoing. Conclusions: We present a novel peptide delivery platform that addresses the challenges inherent to peptide delivery approaches. The presented work demonstrates the feasibility of this platform in delivering Myc inhibitory peptides and provides rationale for further in vivo studies in Myc-dependent tumor models.

SmiA is a hybrid priming/scaffolding adaptor for the LonA protease in Bacillus subtilis

Regulatory proteolysis targets properly folded clients via a combination of cis-encoded degron sequences and trans-expressed specificity factors called adaptors. SmiA of Bacillus subtilis was identified as the first adaptor protein for the Lon family of proteases, but the mechanism of SmiA-dependent proteolysis is unknown. Here, we develop a fluorescence-based assay to measure the kinetics of SmiA-dependent degradation of its client SwrA and show that SmiA–SwrA interaction and the SwrA degron were both necessary, but not sufficient, for proteolysis. Consistent with a scaffolding adaptor mechanism, we found that stoichiometric excess of SmiA caused substrate-independent inhibition of LonA-dependent turnover. Furthermore, SmiA was strictly required even when SwrA levels were high suggesting that a local increase in substrate concentration mediated by the scaffold was not sufficient for proteolysis. Moreover, SmiA function could not be substituted by thermal denaturation of the substrate, consistent with a priming adaptor mechanism. Taken together, we conclude that SmiA functions via a mechanism that is a hybrid between scaffolding and priming models.

Optimizing the cODC degron for Protein Control in Nematode Worms

Caenorhabditis elegans, a nematode worm, is a model multicellular organism for the study of development and disease. Current methods of controlling essential and pleiotropic gene product levels in C. elegans suffer from limitations, including incomplete penetrance, false negatives, and the inability to differentially control multiple genes with the same system simultaneously. Genetic control methods such as loss‐of‐function alleles and CRISPR‐Cas9 targeted genes have proved successful at deducing the relationship between gene function and phenotype; but they lack the ability to investigate genes essential for viability, fertility, and pleiotropic genes that have dissimilar roles in different tissues during the nematode’s lifespan. RNAi is an effective method for studying gene function in C. elegans, it is easy to deliver, can target virtually any sequence, and can be administered at any time during development. However, RNAi suffers from several limitations including: a significant portion of genes cannot be adequately silenced, RNAi cannot enter equally into all tissues, there is a risk of off‐target effects, and the observation that RNAi phenotypes may be delayed if existing proteins persist for long periods of time. Protein degradation methods can overcome many limitations of the genetic methods by genetic fusion of the gene of interest to an inducible protein degradation domain. Some protein control methods lack the ability to target control of protein degradation to specific tissues, while all current methods are limited by the use of a single input signal to control degradation. The overarching goal of this study is to develop a system for complex control of protein function in nematodes using the Latching Orthogonal Cage/Key pRoteins (LOCKR) system, a de novo‐designed protein Switch comprised of a Cage and a Latch that is capable of caging any linear protein sequence. To conditionally control protein levels in nematodes, we will fuse target genes to LOCKR with a cODC degron sequence encoded in the Latch. The interaction of the Cage with the Latch blocks the accessibility of the degron. Introducing a peptide Key releases the Latch, resulting in exposure of the degron sequence and degradation of fused proteins. Here we describe the development of a bacterially‐expressed LOCKR system suitable for in vitro degron optimization. The mEGFP‐degronLOCKR fusion expresses in bacteria and can be purified using immobilized metal affinity chromatography. We hypothesize that when Switch is activated by the Key and the degron is exposed, the mEGFP‐degronLOCKR construct will be degraded in C. elegans lysate. We further predict that various mutations in the degron will affect the rate of target protein degradation. This study will allow more detailed and complex studies of development and disease in C. elegans and may be applicable to other multicellular organisms.

Exploring additional sleep roles for the PERIOD homolog LIN‐42 using the auxin‐inducible degradation system in Caenorhabditis elegans

Sleep has been observed in all animals with a nervous system, however, the neural and molecular mechanisms that regulate it are not fully understood. The genetically tractable nematode Caenorhabditis elegans has a simple and fully mapped nervous system and has emerged as a powerful sleep model. C. eleganssleeps concurrently with each larval molt, called developmentally timed sleep (DTS), which is timed by the PERIOD(PER) homolog LIN‐42. C. elegans also sleep during adulthood in response to cellular damage incurred by stressful stimuli, called stress‐induced sleep (SIS). Based on LIN‐42’s central role in the timing of DTS, we sought to examine its role in the timing of SIS. Null mutations of lin‐42are lethal, so to study the requirement of LIN‐42 during SIS, we used CRISPR/Cas9 to insert the auxin‐induced degron and green fluorescent protein sequence in frame with each isoform of the lin‐42 gene. In the absence of auxin these worms are viable and grow successfully to adulthood. We are currently constructing transgenic strains that will allow us to degrade the LIN‐42 in different subsets of cells. To do this we are expressing the coding sequence for the auxin perceptive F‐box protein TIR1 from different tissue‐specific promoters, including those expressed in all cells, and specific sleep‐regulating neurons. Once these strains are constructed we will degrade LIN‐42 protein and measure the effects on SIS.

Extended JAZ degron sequence for plant hormone binding in jasmonate co-receptor of tomato SlCOI1-SlJAZ

(+)-7-iso-Jasmonoyl-l-isoleucine (JA-Ile) is a lipid-derived phytohormone implicated in plant development, reproduction, and defense in response to pathogens and herbivorous insects. All these effects are instigated by the perception of JA-Ile by the COI1-JAZ co-receptor in the plant body, which in Arabidopsis thaliana is profoundly influenced by the short JAZ degron sequence (V/L)P(Q/I)AR(R/K) of the JAZ protein. Here, we report that SlJAZ-SlCOI1, the COI1-JAZ co-receptor found in the tomato plant, relies on the extended JAZ degron sequence (V/L)P(Q/I)AR(R/K)XSLX instead of the canonical JAZ degron. This finding illuminates our understanding of the mechanism of ligand perception by JA-Ile in this plant, and will inform both efforts to improve it by genetic modification of the SlCOI1-SlJAZ co-receptor, and the development of the synthetic agonists/antagonists.

Whole Exome Sequencing Reveals High Genetic Diversity in Relapsed Multiple Myeloma

Background: Treatments like lenalidomide and proteasome inhibitors alone or in combination with chemotherapy have dramatically improved the outcome in multiple myeloma in the past years. Although the majority of patients achieves a remission, almost all patients eventually relapse. Previous studies have found recurrent mutations in TP53 and FAM46C (Weinhold et al., Blood 2016) in relapsed multiple myeloma as well as CRBN mutations (Kortüm et al., Blood 2016) specifically in lenalidomide-treated patients.Methods: In order to identify additional genetic aberrations that are associated with therapy resistance, we analyzed CD138-selected multiple myeloma cells in bone marrow samples obtained pretreatment (n=12) and at different time points at progression during treatment (n=17) by fluorescence in situ hybridization (FISH) and whole exome sequencing (WES). Peripheral blood was used as a germline control. The median number of therapy lines between pretreatment and progression samples was 1 (range, 1 - 4). Progression samples were obtained after treatment with lenalidomide (n=16), bortezomib (n=14), or high-dose melphalan/autologous stem cell transplantation (n=9) alone or in combination or as sequential treatment. WES was performed by paired-end sequencing (read length 100 bp) on an Illumina HiSeq platform. The mean coverage was 117x. A variant allele frequency (VAF) of 5% was set as a cut-off for mutation calling.Results: In the pretreatment samples, the median number of cytogenetic aberrations was 2 (range, 1 - 4) and the median number of somatic variants was 39 (range, 24 - 63) per sample. There was no significant change in these numbers in posttreatment samples: median number of cytogenetic aberrations 2 (range, 0 - 4), median number of somatic variants 52 (range, 11 - 107).The most recurrent cytogenetic alterations at diagnosis were +1q21 (n=6), +9q34.2 (n=4), and del13q14 (n=3). At relapse, 13 of the 18 cytogenetic abnormalities detected at diagnosis were still present, 5 were lost and 5 were newly acquired. The most frequently lost aberration was +9q34.2 (n=2) and the most commonly acquired aberrations were +1q21 (n=2) and del13q14 (n=2). In pretreatment samples we found recurrent mutations in NRAS (n=4), KRAS (n=3), DIS3 (n=3), and IGLL5 (n=3). The median stability of mutations between pre- and posttreatment samples was 62% (range, 13 - 81%). NRAS mutations were lost in 2 progression samples and DIS3 in 1 progression sample while KRAS mutations remained stable. We found 430 relapse-specific mutations in the 17 posttreatment samples. Mutations in TP53 (n=4) were the most frequently newly acquired genetic events in progression samples, which is consistent with previous studies. Of the newly acquired gene mutations, only eight genes were affected in more than one progression sample, all other gene mutations were non-recurrent. Among genes involved in the mechanism of lenalidomide we found a DDB1 mutation in one progression sample after lenalidomide treatment. Another patient had an IKZF3 mutation at relapse. However, the mutation was located outside the lenalidomide degron sequence and the VAF was only 5%, suggesting that it was not responsible for lenalidomide resistance in the major clone. No relapse-specific mutations were found in other genes encoding proteins involved in lenalidomide or proteasome inhibitor activity like IKZF1, IRF4, CRBN, CUL4A, CUL4B or proteasome subunits. Pathway analyses revealed that mutations in genes involved in WNT/beta catenin and KRAS signaling were enriched in the progression samples.Conclusions: In conclusion, we found a high degree of genetic heterogeneity in relapsed multiple myeloma. There were no obvious mutations in genes involved in lenalidomide or proteasome activity that might drive drug resistance. TP53 mutations and +1q21 were the most recurrent gene aberrations identified at relapse, however, these events may cause a general robustness of multiple myeloma cells to multiple treatment modalities and might not be treatment specific. These results suggest that the genetic basis for drug resistance is heterogeneous and warrants genetic characterization of further patients by DNA and RNA sequencing as well as functional analyses of candidate genes and pathways. DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Does Saccharomyces cerevisiae Require Specific Post-Translational Silencing against Leaky Translation of Hac1up?

HAC1 encodes a key transcription factor that transmits the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus and regulates downstream UPR genes in Saccharomyces cerevisiae. In response to the accumulation of unfolded proteins in the ER, Ire1p oligomers splice HAC1 pre-mRNA (HAC1u) via a non-conventional process and allow the spliced HAC1 (HAC1i) to be translated efficiently. However, leaky splicing and translation of HAC1u may occur in non-UPR cells to induce undesirable UPR. To control accidental UPR activation, multiple fail-safe mechanisms have been proposed to prevent leaky HAC1 splicing and translation and to facilitate rapid degradation of translated Hac1up and Hac1ip. Among proposed regulatory mechanisms is a degron sequence encoded at the 5′ end of the HAC1 intron that silences Hac1up expression. To investigate the necessity of an intron-encoded degron sequence that specifically targets Hac1up for degradation, we employed publicly available transcriptomic data to quantify leaky HAC1 splicing and translation in UPR-induced and non-UPR cells. As expected, we found that HAC1u is only efficiently spliced into HAC1i and efficiently translated into Hac1ip in UPR-induced cells. However, our analysis of ribosome profiling data confirmed frequent occurrence of leaky translation of HAC1u regardless of UPR induction, demonstrating the inability of translation fail-safe to completely inhibit Hac1up production. Additionally, among 32 yeast HAC1 surveyed, the degron sequence is highly conserved by Saccharomyces yeast but is poorly conserved by all other yeast species. Nevertheless, the degron sequence is the most conserved HAC1 intron segment in yeasts. These results suggest that the degron sequence may indeed play an important role in mitigating the accumulation of Hac1up to prevent accidental UPR activation in the Saccharomyces yeast.

A new functional JAZ degron sequence in strawberry JAZ1 revealed by structural and interaction studies on the COI1\u2013JA-Ile/COR\u2013JAZs complexes

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes as developmental and defense responses. JA-Ile mediates the interaction between the F-box protein COI1 (part of the SCFCOI1 E3 ubiquitin ligase) and a JAZ repressor leading to early jasmonate responses. The Arabidopsis JAZ1 protein contains the canonical LPIARR degron sequence, which is responsible for the stabilization of the AtCOI1-JA-Ile-AtJAZ1 complex. In strawberry (Fragaria × ananassa) JAZ family was described at the transcriptional level during fruit development but the information about the interaction mode of this complex is still scarce at the molecular level. To gain insight into the strawberry JA-Ile receptor complex, we evaluated the interaction at the structural level, and protein models were built and analyzed for FaCOI1 and FaJAZ1, FaJAZ8.1, and FaJAZ10. The interaction between FaCOI1 and FaJAZ1, FaJAZ8.1 and FaJAZ10 were explored using several ligands, through molecular docking and molecular dynamics (MD) simulations, finding the strongest interaction with (+)-7-iso-JA-Ile than other ligands. Additionally, we tested interactions between FaCOI1 and FaJAZs by yeast two-hybrid assays in the presence of coronatine (COR, a JA-Ile mimic). We detected strong COR-dependent interactions between FaCOI1 and FaJAZ1. Interestingly, FaJAZ1 contains a new non-canonical (IPMQRK) functional degron sequence, in which Arg and Lys are the key residues for maintaining the interaction of the FaCOI1–COR–FaJAZ1 complex as we observed in mutated versions of the FaJAZ1 degron. Phylogenetic analysis showed that the IPMQRK degron is only present in orthologs belonging to the Rosoideae but not in other Rosaceae subfamilies. Together, this study uncovers a new degron sequence in plants, which could be required to make an alternative and functional JA-Ile perception complex in strawberry.

Evolutionary Analysis of JAZ Proteins in Plants: An Approach in Search of the Ancestral Sequence.

Jasmonates are phytohormones that regulate development, metabolism and immunity. Signal transduction is critical to activate jasmonate responses, but the evolution of some key regulators such as jasmonate-ZIM domain (JAZ) repressors is not clear. Here, we identified 1065 JAZ sequence proteins in 66 lower and higher plants and analyzed their evolution by bioinformatics methods. We found that the TIFY and Jas domains are highly conserved along the evolutionary scale. Furthermore, the canonical degron sequence LPIAR(R/K) of the Jas domain is conserved in lower and higher plants. It is noteworthy that degron sequences showed a large number of alternatives from gymnosperms to dicots. In addition, ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs are displayed in all plant lineages from liverworts to angiosperms. However, the cryptic MYC2-interacting domain (CMID) domain appeared in angiosperms for the first time. The phylogenetic analysis performed using the Maximum Likelihood method indicated that JAZ ortholog proteins are grouped according to their similarity and plant lineage. Moreover, ancestral JAZ sequences were constructed by PhyloBot software and showed specific changes in the TIFY and Jas domains during evolution from liverworts to dicots. Finally, we propose a model for the evolution of the ancestral sequences of the main eight JAZ protein subgroups. These findings contribute to the understanding of the JAZ family origin and expansion in land plants.