Substrate Enrichment Research Articles

Bioorthogonal Monomycolate of Trehalose Disclosed the O-Mycoloylation of Mycoloyltransferases and Other Cell Envelope Proteins in C. glutamicum.

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Many questions about the specificity of protein O-mycoloylation remain crucial for understanding its evolutionary significance in Mycobacteriales and its role in cell physiology. We have developed the first bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct, unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ΔmytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

Hydrophobic Ionic Liquid Gel Microspheres as Bi‐Component Carriers with a Liquid Phase to Immobilize Enzymes for Enhanced Performance

AbstractThis study focuses on incorporating liquid molecules, different from the bulk solution, into the immobilized enzyme carrier to regulate the distribution effect and diffusion‐limiting impact of the carrier's microenvironment for substrates, which generally is difficult to achieve due to the instability of the materials with liquid inclusions. A freestanding liquid‐holding particle carrier, with bi‐component hydrophobic ionic liquid gel microspheres with poly (glycidyl methacrylate) as the network and 1‐butyl‐3‐methylimidazolium hexafluorophosphate as the dispersing medium, is proposed, which is stable in air aqueous solution, and can extract proteins and organic small molecules into its interior due to the mobility of its dispersing medium. Horseradish peroxidase is covalently immobilized into the microspheres, forming a liquid compartment enzyme microreactor. The microreactor exhibits superior stability, enzymatic activity, and catalytic performance for Basic Orange II degradation compared to free enzyme and liquid‐free immobilized enzymes. This is attributed to the biocompatibility of the ionic liquid, its role in substrate enrichment in its interior, and its rapid mass transfer capability. This contribution shows the effectiveness of regulating the carrier's microenvironment with liquid molecules, offering fresh perspectives and strategies for enzyme technology.

Valorization of Fecal Sludge Stabilization via Vermicomposting in Microcosm-Enriched Substrates Using Organic Plant-Derived Materials and Agricultural Soil for Compost Production

The enormous generation of fecal sludge without proper treatment is a major sanitation problem. Vermicomposting of fecal sludge is an innovative way of producing value added compost called vermicompost. A vermicomposting technique that involves augmenting vermibed substrates with organic rich materials to provide additional nutrients and underlying layers needed for microcosm development to produce desirable vermicompost is mostly referred to as substrate enrichment. This study investigated the substrate enrichment of using plant-derived materials, which include coconut coir, palm coir and saw dust, combined with agricultural soil for the production of vermicompost from fecal sludge. Enriched substrates were prepared with 80 g of agricultural soil as amending material, 120 g of fecal matter (65-70% dry matter) and 160 g of the plant-derived materials. The treatments were labeled T1-T3, thus, T1 contained coconut coir, T2 contained palm coir and T3 contained saw dust, followed by a control treatment (T4) that had no plant-derived material. Treatments were triplicated and about 3-week-old 20 clitellated, E. fetida (live weight ∼255–275 mg) were used in the vermicomposting for 12 wk. Physicochemical parameters such as pH, Organic Carbon (Corg), Total Nitrogen (Ntot), Available Phosphorus (Pavail), Exchangeable Calcium (Caexch), Iron (Fe), Lead (Pb) and Aluminum (Al) test were performed. Vermicompost at T1 had the most Corg mineralization, most enhancement in Ntot, Pavail, Caexch, and least concentration of Fe, Pb and Al. Less than 20% earthworm mortality was recorded in all treatments except T4 (28.38 ± 8.71%). The enriched substrates were suitable for vermiculture with a worm weight ranged from 593.77 to 569.39 ± 2.30 mg and the vermicompost recorded high nutrient contents. The nutritious nature and the reduced metal concentration of the produced vermicompost might be safe and productive for agricultural use. However, further studies on the effectiveness of the vermicompost on plants growth and yield needs to be investigated.

Yolk-Shell Au NPs@Carbon Porous Nanoreactors Derived from ZIF-8: A High-Efficiency Catalyst for Three-Component Coupling Reaction.

A yolk-shell Au NPs@carbon porous nanoreactor with an active gold (Au) core and a porous carbon shell has been fabricated and demonstrates excellent high activity and cyclic stability as a heterogeneous catalyst for the three-component coupling reaction of aldehyde, amine, and alkyne. Remarkably, the unique yolk-shell nanostructure can protect gold nanoparticles (Au NPs) from aggregation, allow for efficient mass transport, and benefit substrate enrichment, giving rise to enhanced activity, stability, and recyclability.

Hollow UiO-66-NH2 Encapsulated Pd Catalysts for Highly Selective Hydrogenation of Furfural to Furfuryl Alcohol.

The selective hydrogenation of furfural (FFA) to furfuryl alcohol (FA) is regarded as attractive transformation to achieve the sustainable synthesis of value-added chemicals from biomass resources. However, the conventional supported catalysts are significantly restricted by their narrow pore size, ununiform dispersion and easy leaching or aggregation of catalytic sites. Herein, we designed hollow UiO-66-NH2 as the support to encapsulate Pd nanoparticles (Pd@H-UiO-66-NH2) to achieve the highly active and selective conversion of FFA to FA. Benefiting from the void-confinement effect and substrate enrichment of hollow structure, as well as the surface wrinkles, the as-prepared catalyst Pd@H-UiO-66-NH2 exhibited 96.8 % conversion of FFA with satisfactory selectivity reaching up to 92.4 % at 80 °C, 0.5 MPa H2 in isopropanol solvent within 6 h. More importantly, as-prepared Pd@H-UiO-66-NH2 catalyst exhibited excellent long-term stability, as well as good universality toward a series of hydrogenation of unsaturated hydrocarbons.

Abstract PO3-13-10: Proteogenomic characterization of primary invasive breast tumors from young women compared to matched tumors from older women

Abstract Introduction: Breast cancer (BC) in women < 40 years old accounts for ~5% of BC diagnosed in the U.S. However, young women have more aggressive tumors and worse outcomes, including higher rates of recurrence and lower disease-free and overall survival, compared to older women. To better understand mechanisms for these disparities, an integrated proteogenomic study of tumors from young (< 40) and older (≥ 60) women was performed through the Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) program. Methods: 34 pairs of retrospectively collected, untreated primary breast tumors from young and older women, matched by immunohistochemistry (IHC) subtype and race, were selected from the Clinical Breast Care Project. Median patient follow-up was 7 years. Tumor cells were enriched by laser microdissection and analyzed using RNA sequencing, whole genome sequencing, global proteomics, phosphoproteomics, and reverse phase protein array (RPPA). Molecular and clinical data from tumors from young and old patients in the TCGA-BC and METABRIC studies, matched by IHC subtype and race, were used to corroborate findings from our cohort. Results are presented comparing young women and their tumors to their older counterparts. Results: Young women have worse clinical outcome measured by progression-free interval. Despite matching for IHC subtypes, tumors from young women are enriched for intrinsic basal-like subtype, BRCA1/2 germline mutations, and had fewer invasive lobular BCs. Differentially enriched molecules and pathways: A number of significantly differentially expressed genes (DEGs) and proteins were detected, which were able to separate luminal subtypes of BC by patient age. IRS1 and IRS2 were enriched at both transcript and protein levels; IRS1 has been reported to promote tamoxifen resistance. We observed limited overlap of DEGs between the APOLLO, TCGA, and METABRIC datasets. In contrast, 14 of the 28 significantly upregulated Cancer Hallmark pathways were also enriched in TCGA-BC and METABRIC datasets; suggesting targeting pathways, vs genes, may be more effective for therapeutic intervention. Integrated pathway/network analysis: Integrated pathway analysis of RNA and protein levels identified 5 Hallmark pathways upregulated in younger women, including ER response and proliferative pathways, and 6 downregulated immune-response pathways; 10 of the 11 were supported by the public datasets. Immune scores were also lower. Kinase substrate enrichment analysis identified 4 kinases with increased activity in tumors from young women. ESR1/ER: We identified for the first time a significant reduction of ER protein expression while confirming previously reported lower ESR1 gene expression and copy number variations. Surprisingly, integrated pathway analysis identified elevation of early and late estrogen responses in young women. ER activation, indicated by phosphorylated ER (pS118) normalized to total ER protein, was elevated in LumA tumors from young women. Conclusions: This first-ever integrated proteogenomic study shows that BC in young women is enriched for more aggressive molecular subtypes and for genes that promote tamoxifen resistance, exhibits lower immune pathways and immune scores, and shows enhanced ER responses despite lower gene and protein expression of ER. These findings may contribute to the understanding of the molecular mechanisms underlying worse outcomes of BC in young women, and offer new insight to therapeutic strategies. Disclaimer: The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DoD or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Jianfang Liu, Anthony Soltis, Nicholas Bateman, Qingrong Chen, Lori Sturtz, Brenda Deyarmin, Mariaelena Pierobon, Tamara Abulez, Anupama Praveen-Kumar, Xijun Zhang, Trinh Nguyen, Chunhua Yan, Ying Hu, Kate Guion, Jeffrey Hooke, Albert Kovatich, Leigh Fantacone-Campbell, Brad Mostoller, Leonid Kvecher, Stella Somiari, Patricia Steeg, Sheila Rajagopal, Kathleen Darcy, Jerry Lee, Clifton Dalgard, Thomas Conrads, Emanuel Petricoin, Daoud Meerzaman, Matthew Wilkerson, APOLLO Research Network, Xiaoying Lin, Craig Shriver, Stanley Lipkowitz, Hai Hu. Proteogenomic characterization of primary invasive breast tumors from young women compared to matched tumors from older women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-10.

Use of Eucalyptus Charcoal Waste in the Formulation of Substrate for the Cultivation of Two Strains (LED 20/11 and LED 20/12) of Lentinula edodes

The shiitake mushroom (Lentinula edodes) is globally valued for its nutrition and medicinal properties. New technologies aim to increase production with less environmental impact, considering materials such as charcoal for substrate enrichment. This manuscript evaluated the effect of fine charcoal (FC) on the substrate formulation of two L. edodes strains (LED 20/11 and LED 20/12). The substrate consisted of 72% eucalyptus sawdust, 12.5% rice bran, 12.5% wheat bran, and 3% calcium carbonate (control treatment without charcoal). Treatments with FC proportionally reduced the use of sawdust, with doses of 1%, 2%, 4%, 8%, and 16% (relative to the substrate material). Yield, mushroom number, and mushroom weight were evaluated. The concentration of FC significantly affected the parameters analyzed, especially at the 4% dose. A negative correlation between mushroom number and weight was observed. For yield, the control treatment and the lowest dose of FC (1%) had the highest yields for the first harvest. Strain LED 20/12 showed lower yield variability due to the percentage of FC applied to the substrate. The incorporation of FC into the substrate for shiitake cultivation demonstrates efficacy; however, both the concentration and strain used are limiting factors for its applicability.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Co-Immobilization of Laccase and Mediator into Fe-Doped ZIF-8 Significantly Enhances the Degradation of Organic Pollutants.

Co-immobilization of laccase and mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for wastewater treatment could simultaneously achieve the reusability of laccase and avoid secondary pollution caused by the toxic ABTS. Herein, Fe-induced mineralization was proposed to co-immobilize laccase and ABTS into a metal-organic framework (ZIF-8) within 30 min. Immobilized laccase (Lac@ZIF-8-Fe) prepared at a 1:1 mass ratio of Fe2+ to Zn2+ exhibited enhanced catalytic efficiency (2.6 times), thermal stability, acid tolerance, and reusability compared to free laccase. ABTS was then co-immobilized to form Lac+ABTS@ZIF-8-Fe (ABTS = 261.7 mg/g). Lac@ZIF-8-Fe exhibited significantly enhanced bisphenol A (BPA) removal performance over free laccase due to the local substrate enrichment effect and improved enzyme stability. Moreover, the Lac+ABTS@ZIF-8-Fe exhibited higher BPA removal efficiency than the free laccase+ABTS system, implying the presence of a proximity effect in Lac+ABTS@ZIF-8-Fe. In the successive malachite green (MG) removal, the MG degradation efficiency by Lac@ZIF-8-Fe was maintained at 96.6% at the fifth reuse with only an extra addition of 0.09 mM ABTS in each cycle. As for Lac+ABTS@ZIF-8-Fe, 58.5% of MG was degraded at the fifth cycle without an extra addition of ABTS. Taken together, this research has provided a novel strategy for the design of a co-immobilized laccase and ABTS system for the degradation of organic pollutants.

A constitutional isomer selective chemical proteomic strategy for system-wide profiling of protein lysine 5-hydroxylation.

Lysine 5-hydroxylation (5-Hyl) has been well recognized as an essential protein post-translational modification regulating cellular structural stability, RNA alternative splicing and epigenetic gene expression. System-wide enrichment and quantification of 5-Hyl targets have been challenging due to their chemical inert nature and difficulties in differentiating structural isomers in a complex biological sample. Here, we report the development of an efficient chemical proteomic workflow for affinity enrichment and constitutional isomer specific profiling of endogenous 5-Hyl substrates based on highly selective periodate chemistry. Our study confidently identified over 1600 5-Hyl sites on 630 proteins in human 293T cells, revealing functional significance of the modification in protein structure, transcription and chromatin regulation. Analysis of histone 5-Hyl sites showed that histones H2B and H1 are major targets of the 5-hydroxylysine epigenetic mark. Quantitative proteomic analysis through our chemical enrichment workflow identified specific 5-Hyl substrate proteins mediated by the overexpression of Jumonji-domain containing protein 6 (JMJD6). Our study uncovered two cancer-relevant alternative splice isoforms of JMJD6 that regulate 5-Hyl proteins in distinct cellular pathways, providing unique insights into the functional roles of JMJD6 alternative splicing in transcriptional regulation and cellular development.

Ultra‐low Mn(salen) supported on lignin and selective epoxidation of olefins

AbstractPrecise design of highly efficient, low metal load and eco‐friendly catalysts is the key to sustainable catalysis and green chemistry. In this work, we develop an ultra‐low loading of Mn(salen) complexes covalent grafted with lignin and applied in the selective epoxidation of olefins using O2 as an oxidant. Lignin not only provides the Mn(salen) graft sites but also promotes substrate enrichment via the π‐stacking interaction between substrate and supports. In particular, high turnover frequencies (TOF) of 1833 h−1 can be obtained for styrene epoxidation under mild conditions, which can be superior to most reported Mn(salen) supported catalysts. Compared with cellulose, the lignin‐based catalysts exhibit a higher selectivity toward styrene oxide during the epoxidation of styrene under mild conditions. Controlled experiments indicate that the synergistic effect between phenolic hydroxyl groups and the Mn(salen) complex can improve the conversion rate and selectivity during the selective epoxidation of styrene. This work designs a series of lignin‐based catalysts with ultra‐low Mn(salen) loading, which broadens the utilization of lignin and gives enlightenment for the rational design of lignin‐based catalysts.

Post-transcriptional capping generates coenzyme A-linked RNA

ABSTRACT NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3’-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4’-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4–10 unpaired nucleotides at the 5’ terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5’ triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5’OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4–10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5’ structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Cardiomyocyte intercellular signalling increases oxidative stress and reprograms the global‐ and phospho‐proteome of cardiac fibroblasts

AbstractPathological reprogramming of cardiomyocyte and fibroblast proteome landscapes drive the initiation and progression of cardiac fibrosis. Although the secretome of dysfunctional cardiomyocytes is emerging as an important driver of pathological fibroblast reprogramming, our understanding of the downstream molecular players remains limited. Here, we show that cardiac fibroblast activation (αSMA+) and oxidative stress mediated by the secretome of TGFβ‐stimulated cardiomyocytes is associated with a profound reprogramming of their proteome and phosphoproteome landscape. Within the fibroblast global proteome there was a striking dysregulation of proteins implicated in extracellular matrix, protein localisation/metabolism, KEAP1‐NFE2L2 pathway, lysosomes, carbohydrate metabolism, and transcriptional regulation. Kinase substrate enrichment analysis of phosphopeptides revealed potential role of kinases (CK2, CDK2, PKC, GSK3B) during this remodelling. We verified upregulated activity of casein kinase 2 (CK2) in secretome‐treated fibroblasts, and pharmacological CK2 inhibitor TBB (4,5,6,7‐Tetrabromobenzotriazole) significantly abrogated fibroblast activation and oxidative stress. Our data provides molecular insights into cardiomyocyte to cardiac fibroblast crosstalk, and the potential role of CK2 in regulating cardiac fibroblast activation and oxidative stress.

An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

Abstract B070: Global proteomics and phosphoproteomics profiling of prostate cancer patient derived xenografts tumors from the LuCaP series

Abstract Castration Resistant Prostate Cancer (CRPC) is a form of prostate cancer (PCa) that is resistant to androgen deprivation therapies. Resistance to these therapies leads to metastatic CRPC of adenocarcinoma (AD) origin and can transform to emergent aggressive variant prostate cancer (AVPC) which has genetic aberration features similar to neuroendocrine (NE) phenotypes. Understanding and identifying the underlying mechanisms of resistance and genetic transformations leading to protein alterations will enable us to develop novel therapeutic methods to counteract this resistance and improve outcomes for men with lethal metastatic prostate cancer. To this end, we used patient-derived xenografts (PDXs), an in-vivo clinically relevant model from advanced prostate cancer (PC) patients, to investigate the biology, identify novel protein targets and evaluate new treatment modalities. We aimed to identify known and novel protein kinase targets that might be phospho-regulated unique between CRPC, in AD and AVPC in NE. To pursue this, we applied a mass spectrometry base phosphoproteomics analysis. We processed 48 PDX samples, which included 15 non-castrated (NCR), 18 castration resistant (CR), which are the AD samples and 15 AVPC, the NE tumors. To enrich for global phosphorylated residues, we used sequential metal oxide affinity chromatography. We sequenced peptides from each sample using liquid chromatography in tandem with Orbitrap Eclipse Tribid MS and FAIMS technology for 2 hour gradients. To normalize and calibrate the LC method and to compare across all 48 runs, we spiked-in 400 femtomol of internal standards (iRT) to each sample. For peptide sequence and search analysis, we used Maxquant search algorithm. Using a &amp;gt; 0.75 probability phospho-residue cut-off and 5% FDR, we identified more than 9,700 phospho-residues. We identified RET, ASCL1, CHGA and SYP in NEPC and Androgen Receptor (AR), FOXA1, HOXB13, NKX3.1 and TACSTD2 in the adenocarcinoma PDX LuCap tumor samples, which confirms that our approach detected specific proteins already previously established. We subsequently performed a kinase substrate enrichment analysis and identified GSK-3, ERK1, ERK2, CDK5 kinases to be significantly enriched in the NE tumors while ATM kinase and PKC alpha and beta were enriched in the AD PDX tumor samples. Hierarchical clustering analysis across NE versus AD tumor samples indicated that samples within each group clustered uniquely where we observed intra and inter sample variability. We generated pathway enrichment and GSEA analysis, identified novel targets for therapeutic treatments and novel surface proteins. In conclusion, we have performed a global mass spectrometry based proteomic analysis and created an important first global phosphoproteomics and proteomics database that provides insights into the differentially regulated protein and phosphoproteins between AD and NE PDX tumors and provide a protein database source to develop new hypothesis. Citation Format: Zoi E. Sychev, Gabbrianne E. Larson, Hannah E. Bergom, Abderrahman E. Day, Megan E. Ludwig, Atef E. Ali, Eva Corey, Stephen R. Plymate, Justin E. Hwang, Justin M. Drake. Global proteomics and phosphoproteomics profiling of prostate cancer patient derived xenografts tumors from the LuCaP series [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B070.

Identification of novel phosphoproteomic biomarkers in patients with advanced hepatocellular carcinoma (HCC).

e15155 Background: HCC is one of the leading causes of cancer deaths worldwide. Despite increasing systemic therapy options for patients with advanced HCC, there are currently no predictive biomarkers to guide therapy selection. Phosphoproteomics has been successfully used to identify accurate predictive biomarkers in other cancers from frozen clinical samples (Dokal et al., 2021 ASCO Annual Meeting). In this study, we test the feasibility of biomarker discovery using phospho- and whole-proteomics analyses on formalin fixed and paraffin embedded (FFPE) tru-cut liver biopsies from patients with HCC. Methods: FFPE tru-cut biopsies (n = 29) were obtained from separate cohorts of 15 HCC patients [tumor (T)] and 14 patients with chronic liver disease with varying levels of cirrhosis, fibrosis or steatosis but without cancer [non-tumor (NT)]. T samples were from advanced HCC patients with varied aetiology (chronic viral hepatitis, alcohol or NAFLD). Proteins were extracted from 10 x 10µm sections; crosslinks reversed before digestion to peptides; and multiple clean-up/enrichment steps before analysis. Peptides were quantified by mass spectrometry and performance assessed using multi-variate and enrichment analyses. Results: Overall, 4978 phosphopeptides and 3721 proteins were quantified across all samples. Multi-variate analyses based on the relative expression of phosphopeptides or proteins showed a clear separation between T and NT samples, as well as phenotype-distinct separation (i.e according to differentiation) within the T group. Subsequent testing also verified that the method could reproducibly measure these HCC-differentiating phosphorylation sites (and proteins). Proteins associated with HCC were regulated as expected in T vs NT, e.g. ASS1 (log2-fold change (FC) = -1.75, p &lt; 0.001), BDH2 (log2-FC = -1.97, p &lt; 0.001). Known drug targets were found to be significantly upregulated in poorly-differentiated HCC, e.g. PARP1 (log2-FC = 1.7, p &lt; 0.001) and AKT1 (log2-FC = 1.5, p &lt; 0.001), as well as previously described phosphorylation sites, e.g. p-CDK1 T14 (log2-FC = 5.95, p = 0.007) and p-MCM2 S139 (log2-FC = 2.98, p = 0.046). Kinase substrate enrichment analysis (Casado et al., Science Signaling 2013) showed increased activity for several kinases in T vs NT, including A-RAF (z-score = 3.2, p &lt; 0.001), MAPK3 (z-score = 2.1, p = 0.022) and TNK2 (z-score = 2.1, p = 0.003). Conclusions: We successfully adapted (phospho-)proteomics to FFPE tru-cut biopsy specimens, enabling reproducible identification of novel and known (phospho-)proteins that distinguished differentiation status. A prospective study utilising this phosphoproteomic analysis pipeline is now underway, recruiting patients with advanced HCC receiving first line standard of care therapy with immune checkpoint inhibitor combinations or tyrosine kinase inhibitors to identify potential predictive biomarkers of response.

Abstract B034: Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers

Abstract Direct inhibitors of mutationally activated KRAS are currently under intense preclinical and clinical development, with one KRASG12C mutant-selective inhibitor approved. However, treatment-associated resistance to KRASG12C inhibitors has been reported, with ~60% of relapsed patients acquiring mutations in signaling components both upstream and downstream, and at the level of RAS itself, that led to reactivation of RAF-MEK-ERK and PI3K-AKT effector pathways. Unexpectedly, we found that ectopic expression of constitutively activated MEK1, ERK1 or ERK2, but not AKT1, drove near-complete resistance to direct inhibitors of KRASG12C or KRASG12D in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). Despite comprehensive studies, how ERK supports KRAS-dependent cancer growth remains poorly understood. Therefore, we focused on delineating the ERK-dependent phosphoproteome. We treated a panel of six KRAS-mutant PDAC cell lines acutely (1 h) and long-term (24 h) with the highly selective ERK1/2 inhibitor SCH772984. Our proteomics analyses identified 5,117 phosphosites on 2,252 proteins significantly dysregulated by ERK inhibition. We first compared our PDAC ERK phosphoproteome with a recent compendium of published direct/indirect ERK substrates identified in non-PDAC cells and found, surprisingly, only 12% overlap. Thus, our dataset substaintially extends the complexity of ERK-regulated phosphoproteins, in part, reflecting a pancreatic cancer selective profile. To elucidate the kinase network that drives our PDAC ERK-dependent phosphoproteome, we utilized our newly described global atlas of kinase-substrate specificities and reported kinase-substrate interaction datasets. This revealed a highly dynamic kinome dominated by ERK at 1 h, then dominated by ERK-regulation of CDK1-6 cell cycle components and RHO GTPase signaling. Next, we established the subcellular distribution of the PDAC ERK phosphoproteome, then evaluated data from DepMap, and found enrichment of nuclear ERK substrates that displayed strong dependencies in PDAC. We also determined the KRASG12C-regulated phosphoproteome in pancreatic, lung, and colorectal cancer cells using the KRASG12C selective inhibitor MRTX1257. We used our global atlas of kinase-motif specificities and ERK-regulated phosphoproteome to determine KRAS-regulated signaling pathways in KRASG12C mutant cancers. We found a high correlation between KRAS- and ERK-regulated phosphorylations, primarily driven by CDK substrates. However, we also noted key differences, principally in DNA damage response pathways not identified in our ERK-regulated phosphoproteome. Finally, we performed a high-throughput drug sensitivity and resistance screen of (DSRT) comprised of &amp;gt;500 clinically actionable drugs in combination with a KRASG12C inhibitor, and found ERK inhibitors as top hits. In summary, our studies establish a comprehensive profile of KRAS-ERK signaling output that may define new therapeutic targets for targeting KRAS- and ERK-dependent cancer growth. Citation Format: Jennifer E. Klomp, Jeff A. Klomp, Nathaniel J. Diehl, Cole A. Edwards, Kristina Drizyte-Miller, Priya S. Hibshman, Runying Yang, Alexis J. Morales, Khalilah E. Taylor, Mariaelena Pierobon, Emanuel F. Petricoin III, Johnson L. Jared, Emily M. Huntsman, Tomer M. Yaron, Markus Vähä-Koskela, Laura E. Herring, Alex W. Prevatte, Natalie K. Barker, Lee M. Graves, Wennerberg Krister, Lewis C. Cantley, James G. Christensen, Adrienne D. Cox, Channing J. Der, Clint A. Stalnecker. Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B034.

Effect of Enriched Substrate on the Growth of the Sea Cucumber Holothuria arguinensis Koehler and Vaney, 1906 Juveniles

The sea cucumber Holothuria arguinensis (Koehler and Vaney, 1906) presents an increasing commercial value in Asian markets and its exportation depends exclusively on wild stock harvesting. Production through aquaculture has been acknowledged as an alternative to supply demand and reduce pressure on natural stocks, but there are still bottlenecks to overcome, such as low growth rate and survival. This study focuses on the evaluation of the growth of juveniles of H. arguinensis through substrate enrichment—direct mixing of Sargassum vulgare with sediment—in recirculating aquaculture systems (RAS), for 4 months, with a baseline diet of Saccorhiza polyschides. Compared to the control (no enrichment), sea cucumbers fed with enriched sediment showed the highest specific growth rate (SGR), the highest growth rate (GR) and the lowest size heterogeneity. The results obtained in this study showed a favorable growth of sea cucumbers when in the presence of a substrate enriched with Sargassum vulgare, representing an important step towards the viability of large-scale sea cucumber rearing in Europe.

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation

IntroductionFuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates.ObjectivesThe goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism.MethodsIsolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles.ResultsPyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation.ConclusionIsotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Modulation of Enzyme Cascade Activity by Local Substrate Enrichment and Exclusion on DNA Nanostructures.

Substrate confinement and channeling play a critical role in multienzyme pathways and are considered to impact the catalytic efficiency and specificity of biomimetic and artificial nanoreactors. Here we reported a modulation of a multienzyme system with the cascade activity impacted by the surface affinity binding to substrate molecules. A DNA origami modified with aptamers was used to bind and enrich ATP molecules in the local area of immobilized enzymes, thereby enhancing the activity of an enzyme cascade by more than 2-fold. Alternatively, DNA nanostructure modified with blocked aptamers does not bind with ATP, thereby reducing the activity of the enzyme cascade. The Michaelis-Menten kinetics showed decreased apparent KM values (∼3-fold lower) for enzyme nanostructures modified with aptamers, suggesting the higher effective substrate concentration near enzymes due to the local enrichment of substrates. Conversely, increased apparent KM values (∼2-fold higher) were observed for enzyme nanostructures modified with blocked aptamers, possibly due to the exclusion of substrates approaching the surface. The similar concept of this modified surface-substrate interaction should be applicable to other multienzyme systems immobilized on nanostructures, which could be useful in the development of biomimetic nanoreactors.