Plants produce diverse secondary metabolites. Although each metabolite is made through its respective biosynthetic pathway, plants coordinate multiple biosynthetic pathways simultaneously. One ...example is an interaction between glucosinolate and phenylpropanoid pathways in
. Glucosinolates are defense compounds made primarily from methionine and tryptophan, while phenylpropanoids are made from phenylalanine. Recent studies have shown that the accumulation of glucosinolate intermediate such as indole-3-acetaldoxime (IAOx) or its derivatives represses phenylpropanoid production
the degradation of phenylalanine ammonia lyase (PAL) functioning at the entry point of the phenylpropanoid pathway. Given that IAOx is a precursor of other bioactive compounds other than glucosinolates and that the phenylpropanoid pathway is present in most plants, we hypothesized that this interaction is relevant to other species.
is an oil crop and produces camalexin from IAOx. We enhanced IAOx production in Camelina by overexpressing
which encodes an IAOx-producing enzyme. The overexpression of
results in increased auxin content and its associated morphological phenotypes in Camelina but indole glucosinolates were not detected in Camelina wild type as well as the overexpression lines. However, phenylpropanoid contents were reduced in
overexpression lines suggesting a link between aldoxime metabolism and phenylpropanoid production. Interestingly, the expression of
was not affected in the overexpression lines although PAL activity was reduced. To test if PAL degradation is involved in the crosstalk, we identified F-box genes functioning in PAL degradation through a phylogenetic study. A total of 459 transcript models encoding kelch-motifs were identified from the
database. Among them, the expression of
involved in PAL degradation is up-regulated in the transgenic lines. Our results suggest a link between aldoxime metabolism and phenylpropanoid production in Camelina and that the molecular mechanism behind the crosstalk is conserved in Arabidopsis and Camelina.
The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction ...conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.
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BackgroundGene-fusion genetic aberrations present unique challenges in cancer diagnosis and management. Current treatment strategies often yield low efficiency due to their non-specific targets ...leading to adverse side effects. Personalized immunotherapies targeting these genetic aberrations can potentially improve therapeutic outcomes. We proposed to create messenger RNA nanoparticles designed to target fusion-driven malignancies, aiming to enhance treatment specificity and minimize classic immunotherapeutic adverse effects.MethodsWe are developing a pipeline to identify gene-fusions, design amplification primers, and classify fusions for treatment using messenger RNA nanoparticles cancer vaccine.1–5 The immunogenicity and safety of this approach are to be evaluated using murine models and spontaneous canine and feline tumors.ResultsWe demonstrated the synthesis of fusion-specific mRNA and identified common fusion breakpoints in various tumor types, such as Ewing sarcoma, glioblastoma, ependymoma, non-small cell lung carcinoma, and clear cell sarcoma. Importantly, we established two primary approaches for our fusion-based messenger RNA nanoparticles: 1) off-the-shelf gene-fusion immunotherapy vaccines, and 2) personalized vaccines developed for rare fusions.ConclusionsPreliminary findings suggest that our formulation can target gene fusions with potentially improved treatment.ReferencesSayour EJ, Grippin A, De Leon G, Stover B, Rahman M, Karachi A, et al. Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy. Nano Lett. 2018.Sayour EJ, De Leon G, Pham C, Grippin A, Kemeny H, Chua J, et al. Systemic activation of antigen-presenting cells via RNA-loaded nanoparticles. OncoImmunology. 2016:e1256527.Sanchez-Perez LA, Choi BD, Archer GE, Cui X, Flores C, Johnson LA, et al. Myeloablative temozolomide enhances CD8(+) T-cell responses to vaccine and is required for efficacy against brain tumors in mice. PLoS One. 2013;8(3):e59082.Mitchell DA, Fecci PE, Sampson JH. Immunotherapy of malignant brain tumors. Immunol Rev. 2008;222:70–100.Badapanda C. Suppression subtractive hybridization (SSH) combined with bioinformatics method: an integrated functional annotation approach for analysis of differentially expressed immune-genes in insects. Bioinformation. 2013;9(4):216–21.Ethics ApprovalAll animal experiments were conducted following protocols approved by the Institutional Animal Care and Use Committee at the University of Florida (protocol number 202009685).
Asparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ...ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1' substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1' pockets of VcAEP, an AEP from
. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1' substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1' substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M
s
, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.
The COVID-19 pandemic has caused about seven million deaths worldwide. Preventative vaccines have been developed including Spike gp mRNA-based vaccines that provide protection to immunocompetent ...patients. However, patients with primary immunodeficiencies, patients with cancer, or hematopoietic stem cell transplant recipients are not able to mount robust immune responses against current vaccine approaches. We propose to target structural SARS-CoV-2 antigens (i.e., Spike gp, Membrane, Nucleocapsid, and Envelope) using circulating human antigen-presenting cells electroporated with full length SARS-CoV-2 structural protein-encoding mRNAs to activate and expand specific T cells. Based on the Th1-type cytokine and cytolytic enzyme secretion upon antigen rechallenge, we were able to generate SARS-CoV-2 specific T cells in up to 70% of unexposed unvaccinated healthy donors (HDs) after 3 subsequent stimulations and in 100% of recovered patients (RPs) after 2 stimulations. By means of SARS-CoV-2 specific TCRβ repertoire analysis, T cells specific to Spike gp-derived hypomutated regions were identified in HDs and RPs despite viral genomic evolution. Hence, we demonstrated that SARS-CoV-2 mRNA-loaded antigen-presenting cells are effective activating and expanding COVID19-specific T cells. This approach represents an alternative to patients who are not able to mount adaptive immune responses to current COVID-19 vaccines with potential protection across new variants that have conserved genetic regions.
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In this study, Ogando-Rivas and colleagues generated SARS-CoV-2 specific T cells using human APCs transduced with full length structural antigen encoding mRNAs. The manufactured T cells also recognized immunogenic hypomutated regions within the Spike gp in healthy donors and COVID-19 recovered donors, suggesting protection against ancient and new variants.
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BackgroundDiffuse midline glioma (DMG) is a universal fatal glial brain cancer in children. We tested our novel multilamellar mRNA lipid particle aggregate vaccine (RNA-LPA, IND19304—Sayour),1 a ...tumor-agnostic treatment platform that encapsulates tumor specific RNA and delivers the payload in a highly immunogenic fashion, as an approach to treating this currently incurable cancer.MethodsUsing the K2 DMG model,2 we implant H3K27M-expressing DMG cells into the 4th ventricle of P1-P3 neonatal C57BL/6 mice. RNA-LPA generated from predicated human H3K27M epitopes or total-tumor mRNA are administered intravenously beginning at day 35. We performed multiparameter 3D geospatial fluorescent microscopy to characterize mRNA transduction. Immunologic responses to treatment were evaluated by multiparameter flow cytometry, microscopy, and cytokine profiling.ResultsMice developed clinical neurological signs of disease by day 30–35. RNA-LPAs targeting human H3K27M epitopes were found to be immunogenic in wild-type mice. Intriguingly, nonspecific enhanced green fluorescent protein (eGFP)-RNA-LPAs resulted in statistically significant survival benefits compared to mice treated with empty LPs. However, tumor-specific RNA-LPAs (either H3K27M-specific or total tumor mRNA-derived) also enhanced survival and additionally resulted in a subset of mice with long-term survival. This survival benefit was observed despite the development of clinical hydrocephalus in mice treated with RNA-LPAs. 3D microscopy established that tumors demonstrated invasive disease and microvascular erosion in mice. We found that mRNA transduces fibroblastic reticular cells (FRCs) in the spleen and lymph nodes, prompting widespread immune activation. Treatment with RNA-LPA led to massive increases in production inflammatory cytokines (i.e. TNF-α) and chemokines (i.e. CCL2), which led to recruitment of the majority of circulating monocytes and lymphocytes to secondary lymphoid organs.ConclusionsRNA-LPAs extend survival in our highly aggressive DMG model, including curative outcomes in cohorts treated with either total tumor or H3K27M RNA-LPs. These data suggest that RNA-LPs are capable of stimulating host adaptive immune responses against established DIPG tumors. Signs of hydrocephalus in treated mice may indicate pseudoprogression due to immunologic response, yet mice were frequently able to survive this development. Future studies will further characterize the immunologic response in these mice and support expansion of our existing IND for a multi-institutional phase I clinical trial for children with DMG, who currently have no curative options.AcknowledgementsWe appreciate funding from the ChadTough Defeat DIPG Foundation and the DIPG/DMG Research Funding Alliance. John Ligon and Elias Sayour contributed equally and are co-senior authors.ReferencesMendez-Gomez H, DeVries A, Castillo P, Stover B, Qdaisat S, Von Roemling C, Ogando-Rivas E, Weidert F, McGuiness J, Zhang D, Chung MC, Li D, Zhao C, Marconi C, Campaneria Y, Chardon-Robles J, Grippin A, Karachi A, Thomas N, Huang J, Milner R, Sahay B, Sawyer WG, Ligon JA, Silver N, Simon E, Cleaver B, Wynne K, Hodik M, Molinaro A, Guan J, Kellish P, Doty A, Lee J-H, Carrera-Justiz S, Rahman M, Gatica S, Mueller S, Prados M, Ghiaseddin A, Mitchell DA, Sayour EJ. mRNA aggregates harness danger response for potent cancer immunotherapy. medRxiv. 2023:2023.03.12.23287108. doi: 10.1101/2023.03.12.23287108.Misuraca KL, Cordero FJ, Becher OJ. Pre-Clinical Models of Diffuse Intrinsic Pontine Glioma. Front Oncol. 2015;5:172. doi: 10.3389/fonc.2015.00172. PubMed PMID: 26258075; PMCID: PMC4513210.Ethics ApprovalWork approved under UF IACUC 202200000375
Verticillium wilt is a severe plant disease that causes massive losses in multiple crops. Increasing the plant resistance to Verticillium wilt is a critical challenge worldwide. Here, we report that ...the hemibiotrophic Verticillium dahliae-secreted Asp f2-like protein VDAL causes leaf wilting when applied to cotton leaves in vitro but enhances the resistance to V. dahliae when overexpressed in Arabidopsis or cotton without affecting the plant growth and development. VDAL protein interacts with Arabidopsis E3 ligases plant U-box 25 (PUB25) and PUB26 and is ubiquitinated by PUBs in vitro. However, VDAL is not degraded by PUB25 or PUB26 in planta. Besides, the pub25 pub26 double mutant shows higher resistance to V. dahliae than the wild-type. PUBs interact with the transcription factor MYB6 in a yeast two-hybrid screen. MYB6 promotes plant resistance to Verticillium wilt while PUBs ubiquitinate MYB6 and mediate its degradation. VDAL competes with MYB6 for binding to PUBs, and the role of VDAL in increasing Verticillium wilt resistance depends on MYB6. Taken together, these results suggest that plants evolute a strategy to utilize the invaded effector protein VDAL to resist the V. dahliae infection without causing a hypersensitive response (HR); alternatively, hemibiotrophic pathogens may use some effectors to keep plant cells alive during its infection in order to take nutrients from host cells. This study provides the molecular mechanism for plants increasing disease resistance when overexpressing some effector proteins without inducing HR, and may promote searching for more genes from pathogenic fungi or bacteria to engineer plant disease resistance.
Peptide asparaginyl ligases (PALs) catalyze transpeptidation at the Asn residue of a short Asn-Xaa1-Xaa2 tripeptide motif. Due to their high catalytic activity toward the P1-Asn substrates at around ...neutral pH, PALs have been used extensively for peptide ligation at asparaginyl junctions. PALs also bind to aspartyl substrates, but only when the γCOOH of P1-Asp remains in its neutral, protonated form, which usually requires an acidic pH. However, this limits the availability of the amine nucleophile and, consequently, the ligation efficiency at aspartyl junctions. Because of this perceived inefficiency, the use of PALs for Asp-specific ligation remains largely unexplored. We found that PAL enzymes, such as VyPAL2, display appreciable catalytic activities toward P1-Asp substrates at pH 4–5, which are at least 2 orders of magnitude higher than that of sortase A, making them practically useful for both intra- and intermolecular ligations. This also allows sequential ligations, first at Asp and then at Asn junctions, because the newly formed aspartyl peptide bond is resistant to the ligase at the pH used for asparaginyl ligation in the second step. Using this pH-controlled orthogonal ligation method, we dually labeled truncated sfGFP with a cancer-targeting peptide and a doxorubicin derivative at the respective N- and C-terminal ends in the N-to-C direction. In addition, a fluorescein tag and doxorubicin derivative were tagged to an EGFR-targeting affibody in the C-to-N direction. This study shows that the pH-dependent catalytic activity of PAL enzymes can be exploited to prepare multifunction protein biologics for pharmacological applications.
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Deubiquitinase-targeting chimeras (DUBTACs) have been recently developed to stabilize proteins of interest, which is in contrast to targeted protein degradation (TPD) approaches that degrade ...disease-causing proteins. However, to date, only the OTUB1 deubiquitinase has been utilized to develop DUBTACs via an OTUB1 covalent ligand, which could unexpectedly compromise the endogenous function of OTUB1 owing to its covalent nature. Here, we show for the first time that deubiquitinase USP7 can be harnessed for DUBTAC development. Based on a noncovalent ligand of USP7, we developed USP7-based DUBTACs that stabilized the ΔF508-CFTR mutant protein as effectively as the previously reported OTUB1-based DUBTAC. Importantly, using two different noncovalent ligands of USP7, we developed the first AMPK DUBTACs that appear to selectively stabilize different isoforms of AMPKβ, leading to elevated AMPK signaling. Overall, these results highlight that, in addition to OTUB1, USP7 can be leveraged to develop DUBTACs, thus significantly expanding the limited toolbox for targeted protein stabilization and the development of novel AMPK DUBTACs as potential therapeutics.
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Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and ...repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.
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