Substrate Specificity Research Articles

Iron oxide-based catalyst of (Fe1-xMgx)O·Fe2O3 with well-dispersed active sites favoring carbon nanotubes growth for high-performance microwave absorption

Traditional catalysts for carbon nanotubes growth require specific substrates to prevent catalyst aggregation, then designing environmentally friendly and efficient catalysts remains enormous challenges. Herein, we developed an iron oxide-based catalyst of (Fe1-xMgx)O·Fe2O3 with catalytic active sites for the low-cost growing of carbon nanotubes (CNT) in chemical vapor deposition (CVD) system. This (Fe1-xMgx)O·Fe2O3 was prepared by solid-phase sintering of iron oxide and magnesium oxide uniformly mixed in mass ratio of 3%∼20 wt% MgO at 1400 °C. The introduction of magnesium oxide possesses some advantages of reducing the decomposition temperature of iron oxide and hindering the aggregation of as-formed Fe catalyst during CVD process. Furthermore, the in-situ growing CNT wrapped up the magnetic (Fe1-xMgx)O·Fe2O3 catalyst to form the 3D CNT/(Fe1-xMgx)O·Fe2O3 with the high-performance of microwave absorption. The maximum absorption of the three-dimensional carbon nanocomposites at 11.8 GHz with a thickness of 2.0 mm is −37.3 dB. The coupling of dielectric loss of CNTs and magnetic loss of (Fe1-xMgx)O·Fe2O3 results in the enhancement of microwave absorption. Therefore, this study provides a low-cost and green route to design the efficient catalyst for carbon nanotubes as well as high-performance absorbing materials.

Diversely regio-oxidative degradation of konjac glucomannan by lytic polysaccharide monooxygenase AA10 and generating antibacterial hydrolysate

Konjac glucomannan (KGM) hydrolysate exhibit various biological activities and health-promoting effects. Lytic polysaccharide monooxygenases (LPMOs) play an important role on enzymatic degradation of recalcitrant polysaccharides to obtain fermentable sugars. It is generally accepted that LPMOs exhibits high substrate specificity and oxidation regioselectivity. Here, a bacteria-derived SmAA10A, with chitin-active with strict C1 oxidation, was used to catalyse KGM degradation. Through ethanol precipitation, two hydrolysed KGM components (4 kDa (KGM-1) and 5 kDa (KGM-2)) were obtained that exhibited antibacterial activity against Staphylococcus aureus. In natural KGM, KGM-1, and KGM-2, the molar ratios of mannose to glucose were 1:2.19, 1:3.05, and 1:2.87, respectively, indicating that SmAA10A preferentially degrades mannose in KGM. Fourier-transform infrared spectroscopy and scanning electron microscopy imaging revealed the breakage of glycosylic bonds during enzymatic catalysis. The regioselectivity of SmAA10A for KGM degradation was determined based on the fragmentation behaviour of the KGM-1 and KGM-2 oligosaccharides and their NaBD4-reduced forms. SmAA10A exhibited diverse oxidation degradation of KGM and generated single C1-, single C4-, and C1/C4-double oxidised oligosaccharide forms. This study provides an alternative method for obtaining KGM degradation components with antibacterial functions and expands the substrate specificity and oxidation regioselectivity of bacterial LPMOs.

FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368–372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.

In vitro demetalation of central magnesium in various chlorophyll derivatives using Mg-dechelatase homolog from the chloroflexi Anaerolineae.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC.The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene.ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB.YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

Structural and functional characterization of sulfurtransferase from Frondihabitans sp. PAMC28461.

Sulfurtransferases transfer of sulfur atoms from thiols to acceptors like cyanide. They are categorized as thiosulfate sulfurtransferases (TSTs) and 3-mercaptopyruvate sulfurtransferases (MSTs). TSTs transfer sulfur from thiosulfate to cyanide, producing thiocyanate. MSTs transfer sulfur from 3-mercaptopyruvate to cyanide, yielding pyruvate and thiocyanate. The present study aimed to isolate and characterize the sulfurtransferase FrST from Frondihabitans sp. PAMC28461 using biochemical and structural analyses. FrST exists as a dimer and can be classified as a TST rather than an MST according to sequence-based clustering and enzyme activity. Furthermore, the discovery of activity over a wide temperature range and the broad substrate specificity exhibited by FrST suggest promising prospects for its utilization in industrial applications, such as the detoxification of cyanide.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

Substrate Selection Criteria in Regulated Intramembrane Proteolysis.

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aβ peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

Abstract 1717: RAS and PP2A co-regulated phosphosite on KDM1A dictates its substrate specificity & transcriptional outcome

Abstract RAS-mediated human cell transformation requires simultaneous inhibition of tumor suppressor phosphatase PP2A; however, the target phosphosites co-regulated by RAS and PP2A are poorly characterized. In this study, we describe the phosphoregulation of an epigenetic regulator, KDM1A, by RAS and PP2A. KDM1A is a lysine-specific demethylase that can act as a transcriptional activator or inhibitor, depending on its specific activity toward various histone marks. However, the post-translational modifications regulating the substrate specificity of KDM1A are poorly addressed. We found that KDM1A physically interacts with PP2A through a conserved binding motif, and that mutating the motif residues decreased its interaction with PP2A. Additionally, PP2A activation or RAS inhibition led to increased chromatin recruitment of KDM1A. This enhanced chromatin recruitment of KDM1A was recapitulated upon CRISPR/CAS9-mediated substitution of the PP2A/RAS-targeted serine to alanine. Using histone proteomics, we further identified that the RAS/PP2A-regulated phosphosite determines the substrate specificity of KDM1A. This is evidenced by an increased demethylation of histone H3K9me2/3, a marker of gene activation. The differential gene expression profile of the phosphorylation mutants of KDM1A predominantly showed increased gene expression, further validating our findings. The gene expression profiles of KDM1A phosphomutant RAS mutant cells demonstrate gene level specificity indicating that this newly discovered KDM1A phosphoswitch has an important role in specifying KDM1A function in cancer. Collectively, our results demonstrate that RAS and PP2A activities converge on phosphoregulation of epigenetic machinery in cancer cells. This RAS and PP2A co-regulated phosphosite on KDM1A dictates both its histone mark specificity and transcriptional outcome. Citation Format: Mukund Sharma, Anna Aakula, Jesse Kamila, Reetta Nätkin, Tiziana Bonaldi, Saverio Minucci, Matti Nykter, Jukka Westermarck. RAS and PP2A co-regulated phosphosite on KDM1A dictates its substrate specificity & transcriptional outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1717.

Abstract 648: Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays

Abstract Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use. However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. We compared the potency of a selection of WEE1 inhibitors when different substrates were used. While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly. Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.

Abstract 5889: An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity

Abstract A hallmark of cancer is the ability to evade immunosurveillance mechanisms and thwart the efficacy of immunotherapeutic agents. The proteasome functions as an essential component of immunosurveillance by generating peptides from intracellular proteins that are then presented to T-cells. Antigens generated by the proteasome promote the infiltration of immune cells into tumors and improve tumor regression in response to immunotherapy. Cancer cells can evade elimination by the immune system by deregulating the antigen presentation machinery to downregulate the expression of proteins recognized by immune cells as antigens, creating an immunosuppressive microenvironment. In contrast to constitutive proteasomes, immunoproteasomes are a highly specialized proteasome variant that are highly expressed in antigen-presenting cells. Immunoproteasomes contain distinct catalytic subunits with active site substrate specificities distinct from constitutive proteasomes. Immunoproteasome degrade intracellular proteins to generate antigenic peptides that are the presented by MHC-class I (MHC-I) molecules on tumor cells for recognition by cytotoxic T-cells. We hypothesized that immunoproteasome activation could concomitantly increase the relative abundance and diversity of MHC-I antigens presented on multiple myeloma (MM) cells. High-throughput screening identified a novel small molecule (Compound A) that selectively increased immunoproteasome catalytic activity. Global proteomic integral stability assays determined that Compound A binds the proteasome structural subunit PSMA1 and that treatment with Compound A increased association of the proteasome activator PA28α/β (PSME1/2) activator with immunoproteasomes. The effect of Compound A on immunoproteasomes was abolished after the ablation of PSMA1, PSME1, or PSME2 or by the treatment of cells with suicide inhibitors that target immunoproteasome-specific catalytic sites. Treatment of MM cells with Compound A doubled the total number of MHC-I-bound peptides and increased the level of specific MHC-bound peptides up to 200-fold. Interestingly, a number of tumor-associated antigens and neoantigens were dramatically unmasked following treatment of MM cells with the immunoproteasome activator. Importantly, the treatment of patient CD138+ cells with Compound A promoted the antimyeloma activity of allogenic and autologous patient-derived CD8+ T-cells. Taken together, our results demonstrate the paradigm-shifting translational impact of immunoproteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets to personalize T-cell-directed immunotherapy. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. An immunoproteasome activator that expands the MHC-class I immunopeptidome, unmasks neoantigens and enhances antimyeloma T-cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5889.

Abstract 4374: PP2A-B56α in nutrient scavenging: Tipping the balance from macropinocytosis to cell death in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths in the US, with the lowest five-year survival rate of all major cancers. Nutrients in the PDAC microenvironment are commonly depleted, with the vital amino acid glutamine among the most deficient metabolites. To circumvent this deprivation, PDAC cells initiate KRAS dependent macropinocytosis, an actin-driven nutrient scavenging pathway. Macropinosomes fuse with lysosomes in a process mediated by the kinase PIKfyve. It allows cells to replenish the nutrients required for survival. As glutamine is essential for PDAC cell survival, therapeutic inhibition of macropinocytosis represents a novel strategy to suppress nutrient acquisition and drive cell death. Protein phosphatase 2A (PP2A) is a heterotrimeric Serine/Threonine phosphatase known to inhibit downstream targets of the KRAS signaling cascade and is implicated in macropinocytosis regulation. PP2A holoenzyme comprises of A, B and C subunits, where the regulatory B subunit provides substrate specificity to the enzyme. Using high molecular weight TMR-Dextran, we demonstrate that overexpression of PP2A-B56α subunit or pharmacological activation by DT061 promotes macropinosome accumulation in cells. Macropinosome accumulation was caused by the inability of macropinosomes to fuse with lysosomes, leading to cell death. The cell death caused by DT061 was significantly rescued when treated with a Rac family inhibitor. To understand if B56α regulates the process of macropinosome-lysosome fusion, we performed co-immunoprecipitation and established the interaction of B56α with PIKfyve. Further, pharmacological PIKfyve inhibition with Apilimod phenocopies DT061 induced vesicle accumulation in cells, suggesting that B56α likely inhibits PIKfyve to promote aberrant macropinocytosis. Prevention of lysosomal fusion will impact nutrient uptake through macropinocytosis. Our metabolomics study confirmed the deprivation of glutamine with activation of PP2A-B56α. This regulation can be therapeutically leveraged to metabolically stress cells with a combination of DT061 and glutamine transport inhibitor V-9302. Combination drug treatment was found to be synergistic in a panel of PDAC cells. Together, these findings indicate that activation of PP2A in late stage PDAC promotes aberrant macropinocytosis by preventing lysosomal fusion through PIKfyve regulation and establishes a novel role of PP2A-B56α in nutrient scavenging and cell death. These pathways can be further exploited to identify potential combination therapeutics in PDAC. Citation Format: Garima Baral, Claire M. Pfeffer, Indiraa Doraivel, Sara N. Filippelli, Brittany L. Allen-Petersen. PP2A-B56α in nutrient scavenging: Tipping the balance from macropinocytosis to cell death in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4374.

Chemical range recognized by the ligand-binding domain in a representative amino acid-sensing taste receptor, T1r2a/T1r3, from medaka fish.

Taste receptor type 1 (T1r) proteins are responsible for recognizing nutrient chemicals in foods. In humans, T1r2/T1r3 and T1r1/T1r3 heterodimers serve as the sweet and umami receptors that recognize sugars or amino acids and nucleotides, respectively. T1rs are conserved among vertebrates, and T1r2a/T1r3 from medaka fish is currently the only member for which the structure of the ligand-binding domain (LBD) has been solved. T1r2a/T1r3 is an amino acid receptor that recognizes various l-amino acids in its LBD as observed with other T1rs exhibiting broad substrate specificities. Nevertheless, the range of chemicals that are recognized by T1r2a/T1r3LBD has not been extensively explored. In the present study, the binding of various chemicals to medaka T1r2a/T1r3LBD was analyzed. A binding assay for amino acid derivatives verified the specificity of this protein to l-α-amino acids and the importance of α-amino and carboxy groups for receptor recognition. The results further indicated the significance of the α-hydrogen for recognition as replacing it with a methyl group resulted in a substantially decreased affinity. The binding ability to the protein was not limited to proteinogenic amino acids, but also to non-proteinogenic amino acids, such as metabolic intermediates. Besides l-α-amino acids, no other chemicals showed significant binding to the protein. These results indicate that all of the common structural groups of α-amino acids and their geometry in the l-configuration are recognized by the protein, whereas a wide variety of α-substituents can be accommodated in the ligand binding sites of the LBDs.

Abstract 137: Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma

Abstract Tumor cells of almost all patients with fibrolamellar carcinoma (FLC) have a somatic mutation, a ~400 kB deletion on one copy of chromosome 19 that results in a fusion transcript of DNAJB1 to PRKACA, the catalytic subunit of protein kinase A. The resulting fusion oncoprotein, DNAJB1::PRKACA, has kinase activity, and is required and sufficient for transformation. To understand the oncogenesis of this tumor and to help identify potential therapeutic targets, we explored the pathways of pathogenesis mediated by the aberrant kinase. We explored three mechanisms that might explain transformation by the oncogenic kinase: - A difference in the substrate specificity of DNAJB1::PRKACA kinase versus native PRKACA kinase. - A difference in the interactome of DNAJB1::PRKACA kinase versus native PRKACA kinase. - An increase of total catalytic kinase activity (DNAJB1::PRKACA + PRKACA) versus the native PRKACA condition. We found, comparing cytosolic extracts of tumor and normal liver cells, that there are differences in the substrate specificity of the DNAJB1::PRKACA kinase. Additionally, such comparisons revealed differences in the interactions between the catalytic subunit of the kinase and cytosolic components. We also demonstrated a consistent increase in total PRKACA catalytic subunit in the FLC tumor, relative to the adjacent nontransformed tissue. Further, there is an increase in free, unregulated catalytic activity in the tumor cells and a change in the localization of the catalytic subunit in the tumor cells. Additionally, we have found that this increase in free catalytic activity is sufficient to account for the changes in the transcriptome in tumor cells of FLC patients. Importantly, enhanced expression in hepatocytes of either DNAJB1::PRKACA kinase or native PRKACA kinase caused very similar changes in the transcriptome, and the resultant transcriptomes closely resembled that of tumor cells from FLC patients. This suggests that the DNAJB1 domain is dispensable and not critical for transformation. In our investigation of patient tumors we found two rare and different subclasses of FLC that are indistinguishable from “classic” FLC, based on their transcriptome and based on their drug response profiles. Importantly, both subclasses are associated with changes of PRKACA, but neither involves the DNAJB1 domain. Thus, the DNAJB1 domain is not a critical component of transformation, and the significant driver is an increase of the cellular catalytic activity. Citation Format: Mahsa Shirani, Michael Tomasini, Solomon Levin, soeren heissel, Henrik Molina, Ype De Jong, charles Rice, Philip Coffino, Barbara Lyons, Sanford Simon. Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 137.

Abstract 3345: PP1 phosphatase complex at the hub of the integrated stress response: A potential therapeutic target in multiple myeloma

Abstract Protein phosphatase-1 (PP1) is a class of eukaryotic enzyme that catalyzes more than half of the phosphoserine/threonine dephosphorylation reactions, and plays essential roles in cell division, glycogen metabolism and protein synthesis. The substrate specificity of PP1 holoenzyme complex is mediated by PP1-interacting proteins and its catalytic subunits. Of note, PPP1R15B/CReP and PPP1R15A/GADD34 are regulatory subunits of PP1 complex that regulates global protein synthesis and mRNA translation through eIF2α dephosphorylation. Notably, PPP1R15B is constitutively expressed, whereas the expression of PPP1R15A is stress-inducible. Previous studies showed that multiple myeloma (MM), a hematological malignancy arising from immunoglobulin-secreting plasma cells, is characterized by continual ER stress and high dependency on the unfolded protein response (UPR). The integrated stress response (ISR) activated by eIF2α phosphorylation is the fastest reaction pathway of the UPR; however, its functional role and therapeutic potential in MM remains largely unknown. In this study, we performed gene expression analysis and super-enhancer (SE) profiling in MM cell lines, primary patient samples, normal CD138+ plasma cells and memory B cells. we found that PPP1R15B is highly expressed in MM cells as compared to healthy controls, but did not observe significant upregulation of PPP1R15A in MM cells. In addition, higher expression of PPP1R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. Depletion of PPP1R15B significantly reduced cell viability, proliferation and induced G2/M phase cell cycle arrest. Gene Ontology analysis of downregulated genes in response to PPP1R15B knockdown indicates significant association with the UPR pathway. Immunoblot analysis showed activation of ER stress pro-apoptotic executors PUMA, NOXA, and cleavage of caspase-12 in PPP1R15B knockdown cells. Furthermore, we examined the therapeutic effects of eIF2α dephosphorylation inhibitors, Salubrinal and Raphin1, on MM cells. Raphin1 is a selective inhibitor of PPP1R15B that binds strongly to the PPP1R15B-PP1c complex, whereas Salubrinal inhibits both PPP1R15A-PP1c and PPP1R15B-PP1c complex. Our data showed that both Salubrinal and Raphin1 inhibited MM cell proliferation in a dose-dependent manner, while sparing normal plasma cells. MM cell lines with elevated PPP1R15B expression level were more sensitive to Salubrinal or Raphin1 than those with lower PPP1R15B level. Similar changes in the expression patterns of ISR-related genes were observed in response to eIF2α dephosphorylation inhibitors. Taken together, our study suggests that targeting PPP1R15B-PP1c complex or maintaining eIF2α hyperphosphorylation may serve as a new therapeutic approach in MM, which warrants further investigation. Citation Format: Sinan Xiong, Jianbiao Zhou, Tze King Tan, Sabrina Hui-Min Toh, Tae-Hoon Chung, Takaomi Sanda, Wee Joo Chng. PP1 phosphatase complex at the hub of the integrated stress response: A potential therapeutic target in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3345.

Encapsulation of Enzymes into Hydrophilic and Biocompatible Metal Azolate Framework: Improved Functions of Biocatalyst in Cascade Reactions and its Sensing Applications.

Multiple enzyme-triggered cascade biocatalytic reactions are vital in vivo or vitro, considering the basic biofunction preservation in living organisms and signals transduction for biosensing platforms. Encapsulation of such enzymes into carrier endows a sheltering effect and can boost catalytic performance, although the selection and preparation of an appropriate carrier is still a concern. Herein, focusing on MAF-7, a category of metal azolate framework (MAF) with superiority against the topologically identical ZIF-8, this enzyme@MAF system can ameliorate the sustainability of encapsulating natural enzymes into carriers. The proposed biocatalyst composite AChE@ChOx@MAF-7/hemin is constructed via one-pot in situ coprecipitation method. Subsequently, MAF-7 is demonstrated to exhibit an excellent capacity of the carrier and protection against external factors in the counterpart of ZIF-8 through encapsulated and free enzymes. In addition, detections for specific substrates or inhibitors with favorable sensitivity are accomplished, indicating that the properties above expectation of different aspects of the established platform are successfully realized. This biofunctional composite based on MAF-7 can definitely provide a potential approach for optimization of cascade reaction and enzyme encapsulation.

The Use of Immobilised Enzymes for Lipid and Dairy Processing and Their Waste Products: A Review of Current Progress

The use of edible oils and fats in dairy products is becoming increasingly important in the food industry because of their complementary functional properties. Most of these products are produced using food-grade enzymes as processing aids because processes involving enzymes are considered mild and environmentally friendly for regulatory purposes. The poor stability and recovery of enzymes in their native state limit their performance, and to enhance their activity, stability, and reusability, enzymes are often immobilised—a process that involves attaching them to a solid support. Additionally, immobilisation enables enzymes to selectively target specific substrates or products, making them highly efficient. These features have led to the increased use of immobilised enzymes in dairy and lipid processing and enzymes have been used to produce a broad range of products such as whey protein concentrates and isolates, peptide–lipid conjugates, lipid concentrates, structured lipids, and human milk fat substitutes. Therefore, this article reviews the current progress on different enzyme preparations and their use in lipid and dairy processing. It also summarises opportunities in enzyme-catalysed valorisation of dairy and lipid waste streams with the ultimate goals of sustainable food production and reductions in waste.

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1021.