Protective effects and molecular mechanisms of Litopenaeus vannamei treated with l-arginine/l-lysine against myofibrillar proteins oxidation and quality degradation during freeze-thaw cycles.

The storage and processing of Litopenaeus vannamei are often challenged by the freeze-thaw (F-T) cycle phenomenon. This study delved into the influence of pretreatment with l-arginine (Arg) and l-lysine (Lys) on the myofibrillar proteins oxidation and quality of shrimp subjected to F-T cycles. Arg and Lys pretreatment notably improved water-holding capacity (WHC), textural integrity as well as the myofibrillar structure of the shrimps. A lesser reduction in the amounts of immobile and bound water was found in the amino acid-treated groups, and the oxidation of lipids and proteins were both decelerated. Molecular simulation results indicated that Arg and Lys could form hydrogen and salt-bridge bonds with myosin, enhancing the stability of Litopenaeus vannamei. The study concludes that Arg and Lys are effective in alleviating the adverse effects of F-T cycles on the quality of Litopenaeus vannamei, and provides a new solution for the quality maintenance during storage and processing.

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.

Effects of dietary digestible lysine levels in breeding Japanese quails on productive and reproductive performance, egg quality, blood metabolites and immune responses.

BACKGROUND: The vegetable-based diet alone does not provide the lysine (Lys) needed to maximize poultry productive performance. OBJECTIVES: This experiment aimed to evaluate the effects of dietary digestible Lys (dLys) level on productive and reproductive performance, egg quality, blood metabolites and immune responses in breeding Japanese quails (Coturnix japonica). METHODS: The experiment was conducted in a completely randomized design with 6 treatments, 5 replicates and 15 (12 females and 3 meals) 10-week-old breeding Japanese quails each. A basal diet was formulated to meet nutritional requirements of breeding quails except dLys. The basal diet was supplemented with graded (+0.82 g/kg) levels of l-Lys-HCl, corresponding to dietary dLys levels of 0.690%, 0.755%, 0.820%, 0.885%, 0.950% and 1.015%. The experiment lasted for 12 weeks, which was divided into 3-4-week periods. RESULTS: Significant differences were observed for egg production (EP), egg mass (EM) and feed efficiency (FE) in response to increasing dietary dLys concentration with quadratic trends. The highest traits were observed in the birds fed with a diet containing 0.885% dLys. However, feed intake, egg quality, reproductive performance, blood metabolites and immune responses against sheep red blood cell inoculation were not significantly affected by increasing dietary dLys concentrations. The dLys requirements during 11-14, 15-18, 19-22 and 11-22 (overall) weeks of age for optimal EP, EM and FE, based on the quadratic broken-line regression analysis, were estimated 272, 265, 250 and 266; 293, 285, 264 and 279; and 303, 294, 281 and 293 mg/bird/day, respectively. CONCLUSIONS: The dLys requirements vary depending on the EP phase and the trait being optimized. The estimated dLys requirement for FE was higher than those for EP and EM. During the peak stage of the first laying cycle, the dietary dLys level of 0.932% and a daily intake of 303 mg dLys/bird are sufficient for optimal performance.

Advanced glycation end-product crosslinking activates a type VI secretion system phospholipase effector protein.

Advanced glycation end-products (AGE) are a pervasive form of protein damage implicated in the pathogenesis of neurodegenerative disease, atherosclerosis and diabetes mellitus. Glycation is typically mediated by reactive dicarbonyl compounds that accumulate in all cells as toxic byproducts of glucose metabolism. Here, we show that AGE crosslinking is harnessed to activate an antibacterial phospholipase effector protein deployed by the type VI secretion system of Enterobacter cloacae. Endogenous methylglyoxal reacts with a specific arginine-lysine pair to tether the N- and C-terminal α-helices of the phospholipase domain. Substitutions at these positions abrogate both crosslinking and toxic phospholipase activity, but in vitro enzyme function can be restored with an engineered disulfide that covalently links the N- and C-termini. Thus, AGE crosslinking serves as a bona fide post-translation modification to stabilize phospholipase structure. Given the ubiquity of methylglyoxal in prokaryotic and eukaryotic cells, these findings suggest that glycation may be exploited more generally to stabilize other proteins. This alternative strategy to fortify tertiary structure could be particularly advantageous in the cytoplasm, where redox potentials preclude disulfide bond formation.

Mechanism of degrader-targeted protein ubiquitinability.

Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.

Epigenetic therapies targeting histone lysine methylation: complex mechanisms and clinical challenges.

As epigenetic therapies continue to gain ground as potential treatment strategies for cancer and other diseases, compounds that target histone lysine methylation and the enzyme complexes represent a major frontier for therapeutic development. Clinically viable therapies targeting the activities of histone lysine methyltransferases (HKMT) and demethylases (HKDMs) have only recently begun to emerge following FDA approval of the EZH2 inhibitor tazemetostat in 2020 and remain limited to compounds targeting the well-studied SET domain-containing HKMTs and their opposing HKDMs. These include the H3K27 methyltransferases EZH2/EZH1, the singular H3K79 methyltransferase DOT1L, and the H3K4 methyltransferase MLL1/COMPASS as well as H3K9 and H3K36 methyltransferases. They additionally include the H3K4/9-preferential demethylase LSD1 and the H3K4-, H3K27-, and H3K36-preferential KDM5, KDM6, and KDM2 demethylase subfamilies, respectively. This Review discusses the results of recent clinical and preclinical studies relevant to all of these existing and potential therapies. It provides an update on advancements in therapeutic development, as well as more basic molecular understanding, within the past 5 years approximately. It also offers a perspective on histone lysine methylation that departs from the long-predominant "histone code" metaphor, emphasizing complex-disrupting inhibitors and proximity-based approaches rather than catalytic domain inhibitors in the outlook for future therapeutic development.

UBR5 mediates colorectal cancer chemoresistance by attenuating ferroptosis via Lys 11 ubiquitin-dependent stabilization of Smad3-SLC7A11 signaling.

Chemoresistance remains a principal culprit for the treatment failure in colorectal cancer (CRC), especially for patients with recurrent or metastatic disease. Deciphering the molecular basis of chemoresistance may lead to novel therapeutic strategies for this fatal disease. Here, UBR5, an E3 ubiquitin ligase frequently overexpressed in human CRC, is demonstrated to mediate chemoresistance principally by inhibiting ferroptosis. Paradoxically, UBR5 shields oxaliplatin-activated Smad3 from proteasome-dependent degradation via Lys 11-linked polyubiquitination. This novel chemical modification of Smad3 facilitates the transcriptional repression of ATF3, induction of SLC7A11 and inhibition of ferroptosis, contributing to chemoresistance. Consequently, targeting UBR5 in combination with a ferroptosis inducer synergistically sensitizes CRC to oxaliplatin-induced cell death and control of tumor growth. This study reveals, for the first time, a major clinically relevant chemoresistance mechanism in CRC mediated by UBR5 in sustaining TGFß-Smad3 signaling and tuning ferroptosis, unveiling its potential as a viable therapeutic target for chemosensitization.

Potential functions and mechanisms of lysine crotonylation modification (Kcr) in tumorigenesis and lymphatic metastasis of papillary thyroid cancer (PTC).

OBJECTIVES: To examine the putative functions and mechanisms of lysine crotonylation (Kcr) during the development and progression of papillary thyroid cancer (PTC). METHODS: Samples of thyroid cancer tissues were collected and subjected to liquid chromatography-tandem mass spectrometry. Crotonylated differentially expressed proteins (DEPs) and differentially expressed Kcr sites (DEKSs) were analyzed by Motif, dynamic expression model analysis (Mfuzz), subcellular localization, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, Go Ontology (GO) annotation, and protein-protein interaction analysis (PPI). Validation was performed by immunohistochemistry (IHC). RESULTS: A total of 262 crotonylated DEPs and 702 DEKSs were quantitated. First, for the tumor/normal comparison, a dynamic expression model analysis (Mfuzz) of the DEKSs revealed that clusters 1, 3, and 4 increased with the progression of thyroid cancer; however, cluster 6 showed a dramatic increase during the transition from N0-tumor to N1-tumor. Furthermore, based on GO annotation, KEGG, and PPI, the crotonylated DEPs were primarily enriched in the PI3K-Akt signaling pathway, Cell cycle, and Hippo signaling pathway. Of note, crosstalk between the proteome and Kcr proteome suggested a differential changing trend, which was enriched in Thyroid hormone synthesis, Pyruvate metabolism, TCA cycle, Cell cycle, and Apoptosis pathways. Similarly, for the LNM comparison group, the DEKSs and related DEPs were primarily enriched in Hydrogen peroxide catabolic process and Tight junction pathway. Finally, according to The Cancer Genome Atlas Program (TCGA) database, the differential expression of Kcr DEPs were associated with the prognosis of thyroid cancer, indicating the prognostic significance of these proteins. Moreover, based on the clinical validation of 47 additional samples, Kcr was highly expressed in thyroid tumor tissues compared with normal tissue (t = 9.792, P < 0.001). In addition, a positive correlation was observed between Kcr and N-cadherin (r = 0.5710, P = 0.0015). Moreover, N-cadherin expression was higher in the relatively high Kcr expression group (χ2 = 18.966, P < 0.001). CONCLUSIONS: Higher Kcr expression was correlated with thyroid tumorigenesis and lymphatic metastasis, which may regulate thyroid cancer progression by Pyruvate metabolism, TCA cycle, Cell cycle, and other pathways.

Chemo mechanical caries removal CMCR: Efficacy of Carisolv gel in primary molar teeth.

Objectives: To evaluate the efficacy of CariSolv gel with respect to chemo-mechanical caries removal in primary molar teeth. METHODS: The cross-sectional study was conducted at the Department of Paediatric Dentistry, Bakhtawar Amin Dental College and Hospital, Multan, Pakistan, from July to December 2022, and comprised patients of either gender aged 6-12 years having vital, primary molar teeth with clinical and radiographic evidence of carious lesion. Freshly prepared CariSolv gel 0.2 ml to 1.0ml was applied to carious dentine for a minimum of 30 seconds, using chemo-mechanical caries removal hand instruments. The cavity preparation was rinsed and dried. Image caries detector dye was applied by micro brush for 10 seconds. After the cavity preparation was washed and dried, any red-stained dentine indicated residual infected dentine. A maximum of 3 chemo-mechanical caries removal cycles were allowed. Data was analysed using SPSS 26.0. RESULTS: Of the 134 patients, 74(55.2%) were boys and 60(44.8%) were girls. The overall mean age was 8.55±1.58 years. The procedure was successful in 115(85.8%) cases. Age and gender were not significantly associated with the outcome (p>0.05). CONCLUSIONS: Chemo-mechanical caries removal method using CariSolv gel was found to be a viable alternative to traditional drilling techniques for caries removal in primary molar teeth.

Genetically encoded epigenetic sensors for visualization of H3K9me3, H3K9ac and H3K4me1 histone modifications in living cells.

Post-translational modifications of histones play a crucial role in chromatin structure maintenance and epigenetic regulation. The LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscape) method represents a promising approach for tracking histone modifications. It involves visualization of epigenetic modifications using genetically encoded fluorescent sensors and further analysis of the obtained intranuclear patterns by multiparametric image analysis. In this study, we designed three new red fluorescent sensors-MPP8-Red, AF9-Red and DPF3-Red-for live-cell visualization of patterns of H3K9me3, H3K8ac and H3K4me1, respectively. The observed fluorescent patterns were visually distinguishable, and LiveMIEL analysis clearly classified them into three corresponding groups. We propose that these sensors can be used for live-cell dynamic analysis of changes in organization of three epigenetic types of chromatin.

Rapid diazotransfer for selective lysine labelling.

Azide functionalization of protein and peptide lysine residues allows selective bioorthogonal labeling to introduce new, site selective functionaltiy into proteins. Optimised diazotransfer reactions under mild conditions allow aqueous diazotransfer to occur in just 20 min at pH 8.5 on amino acid, peptide and protein targets. In addition, conditons can be modified to selectively label a single lysine residue in both protein targets investigated. Finally, we demonstrate selective modification of proteins containing a single azidolysine using copper(I)-catalyzed triazole formation.

Biological Relevance of Dual Lysine and N-Terminal Methyltransferase METTL13.

The dual methyltransferase methyltransferase-like protein 13, also referred to as METTL13, or formerly known as FEAT (faintly expressed in healthy tissues, aberrantly overexpressed in tumors), has garnered attention as a significant enzyme in various cancer types, as evidenced by prior literature reviews. Recent studies have shed light on new potential roles for METTL13, hinting at its promise as a therapeutic target. This review aims to delve into the multifaceted biology of METTL13, elucidating its proposed mechanisms of action, regulatory pathways, and its implications in disease states, as supported by the current body of literature. Furthermore, the review will highlight emerging trends and gaps in our understanding of METTL13, paving the way for future research efforts. By contextualizing METTL13 within the broader landscape of cancer biology and therapeutics, this study serves as an introductory guide to METTL13, aiming to provide readers with a thorough understanding of its role in disease phenotypes.

Design, Synthesis, and Biological Evaluation of Lysine-Stapled Peptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor Activity In Vivo.

We introduce novel lysine-stapled peptide inhibitors targeting p53-MDM2/MDMX interactions. Leveraging the model peptides pDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a series of lysine-stapled analogues were designed and synthesized. Through in vitro cell assay screening, two lead compounds, SPDI-48-T1 and SPMI-48-T3, were identified for their excellent antiproliferation activity. Fluorescence polarization assays revealed that both compounds exhibited strong binding affinities against MDM2 and MDMX, ascertained by Kd values within the low micromolar spectrum. Further characterization of SPDI-48-T1 and SPMI-48-T3 demonstrated that SPDI-48-T1 possessed superior cell permeability and serum stability. Notably, SPDI-48-T1 displayed a dose-dependent suppression of tumor growth in an HCT116 xenograft mouse model. Our findings indicate that SPDI-48-T1 holds promise as a lead compound for further development as an anticancer agent by modulating p53-MDM2/MDMX interactions. Additionally, this study also proved that the lysine stapling strategy may serve as a robust approach for generating peptide ligands targeting other protein-protein interactions.

Solvent-Dependent Chemoselectivity Switch to Arg-Lys Imidazole Cross-Links.

Herein, we report a trifluoroethanol-mediated, chemoselective method for the formation of Arg-Lys imidazole cross-links with methylglyoxal and its application in the selective macrocyclization of peptides between Lys and Arg and the late-stage diversification of Lys-containing peptides with guanidine. Our findings highlight the critical role of solvent choice in controlling chemoselectivity, providing valuable insights into solvent-dependent peptide modification.

Current computational tools for protein lysine acylation site prediction.

As a main subtype of post-translational modification (PTM), protein lysine acylations (PLAs) play crucial roles in regulating diverse functions of proteins. With recent advancements in proteomics technology, the identification of PTM is becoming a data-rich field. A large amount of experimentally verified data is urgently required to be translated into valuable biological insights. With computational approaches, PLA can be accurately detected across the whole proteome, even for organisms with small-scale datasets. Herein, a comprehensive summary of 166 in silico PLA prediction methods is presented, including a single type of PLA site and multiple types of PLA sites. This recapitulation covers important aspects that are critical for the development of a robust predictor, including data collection and preparation, sample selection, feature representation, classification algorithm design, model evaluation, and method availability. Notably, we discuss the application of protein language models and transfer learning to solve the small-sample learning issue. We also highlight the prediction methods developed for functionally relevant PLA sites and species/substrate/cell-type-specific PLA sites. In conclusion, this systematic review could potentially facilitate the development of novel PLA predictors and offer useful insights to researchers from various disciplines.

Engineering Pyrrolysine Systems for Genetic Code Expansion and Reprogramming.

Over the past 16 years, genetic code expansion and reprogramming in living organisms has been transformed by advances that leverage the unique properties of pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs. Here we summarize the discovery of the pyrrolysine system and describe the unique properties of PylRS/tRNAPyl pairs that provide a foundation for their transformational role in genetic code expansion and reprogramming. We describe the development of genetic code expansion, from E. coli to all domains of life, using PylRS/tRNAPyl pairs, and the development of systems that biosynthesize and incorporate ncAAs using pyl systems. We review applications that have been uniquely enabled by the development of PylRS/tRNAPyl pairs for incorporating new noncanonical amino acids (ncAAs), and strategies for engineering PylRS/tRNAPyl pairs to add noncanonical monomers, beyond α-L-amino acids, to the genetic code of living organisms. We review rapid progress in the discovery and scalable generation of mutually orthogonal PylRS/tRNAPyl pairs that can be directed to incorporate diverse ncAAs in response to diverse codons, and we review strategies for incorporating multiple distinct ncAAs into proteins using mutually orthogonal PylRS/tRNAPyl pairs. Finally, we review recent advances in the encoded cellular synthesis of noncanonical polymers and macrocycles and discuss future developments for PylRS/tRNAPyl pairs.

RNF39 facilitates antiviral immune responses by promoting K63-linked ubiquitination of STING.

The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-dependent pathway is a key DNA-sensing pathway that recognizes cytosolic DNA and plays a crucial role in initiating innate immune responses against pathogenic microbes and cancer. Various molecules have been identified as regulators of the cGAS-dependent pathway that controls innate immune responses. However, despite the important roles of Stimulator-of-interferon genes (STING) in the cGAS-dependent pathway, the regulation of its activation has not been elucidated. Here, we show that the E3 ubiquitin ligase, RING finger protein 39 (RNF39), interacts with STING in macrophages and HERK293T cells. Moreover, RNF39 accelerates DNA-sensing pathways by promoting lysine (K)63-linked ubiquitination of STING, and then facilitating the formation of STING-TBK1 complex. Concordantly, Rnf39 deficiency inhibits innate immune responses triggered by DNA viral infection and accelerates viral replication. Furthermore, herpes simplex virus-1 (HSV-1) infection induces RNF39 expression in an IFN-I-dependent manner. Thus, we outline a novel mechanism for controlling STING activation and a feedback mechanism for controlling antiviral immune responses. RNF39 could be a priming intervention target for the prevention and treatment of viral diseases, especially DNA viral infections.

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells.

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Effects of dietary indispensable amino acid deficiencies on feed intake in stomachless fish.

Evidence suggests that fish are more tolerant than mammals to imbalanced dietary amino acid profiles. However, the behavioral and physiological responses of fish to individual deficiencies in dietary indispensable amino acids (IDAA) remain unclear. This study examined how stomachless fish respond to diets deficient in limiting IDAA (lysine, methionine, and threonine), using Zebrafish (Danio rerio) as a model. The response to deficient diets was assessed based on; 1) growth performance and feeding efficiency; 2) feed intake; 3) expression of appetite-regulating hormones and nutrient-sensing receptors; and 4) muscle postprandial free amino acid (FAA) levels. There were 6 treatments, each with 3 replicate tanks. A semi-purified diet was formulated for each group. The CG diet was based on casein and gelatin, while the FAA50 diet had 50 % of dietary protein supplied with crystalline amino acids. Both were formulated to contain matching, balanced amino acid profiles. The remaining diets were formulated the same as the FAA50 diet, with minor adjustments to create deficiencies in selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the FAA mix, respectively, and the Def diet was deficient in all three. The juvenile Zebrafish were fed to satiation 3 times daily from 21 to 50 days-post-hatch. Results showed that 50 % replacement of dietary protein with crystalline amino acids significantly reduced growth of juvenile Zebrafish. There were no significant differences in growth between the FAA50 group and groups that received deficient diets. The deficiency of singular IDAA did not induce significant changes in feed intake; however, the combined deficiency in the Def diet caused a significant increase in feed intake. This increased feed intake led to decreased feeding efficiency. A significant decrease in feeding efficiency was also observed in the (-) Lys group. There was an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group. Overall, results from this study suggest stomachless fish increase feed intake when challenged with IDAA-deficient diets, and the regulation of NPY might play a role in this response.