Microbial enzymes can be used as processing aids or additives in food and feed industries. Enzymatic detoxification of ochratoxin A (OTA) is a promising method to reduce OTA content. Here, we characterize the full-length enzyme ochratoxinase (AnOTA), an amidohydrolase from Aspergillus niger. AnOTA hydrolyzes OTA and ochratoxin B (OTB) mycotoxins efficiently and also other substrates containing phenylalanine, alanine, or leucine residues at their C-terminal position, revealing a narrow specificity profile. AnOTA lacks endopeptidase or aminoacylase activities. The structural basis of the molecular recognition by AnOTA of OTA, OTB, and a wide array of model substrates has been investigated by molecular docking simulation. AnOTA shows maximal hydrolytic activity at neutral pH and high temperature (65 °C) and retained high activity after prolonged incubation at 45 °C. The reduction of OTA levels in food products by AnOTA has been investigated using several commercial plant-based beverages. The results showed complete degradation of OTA with no detectable modification of beverage proteins. Therefore, the addition of AnOTA seems to be a useful procedure to eliminate OTA in plant-based beverages. Moreover, computational predictions of in vivo characteristics indicated that AnOTA is neither an allergenic nor antigenic protein. All characteristics found for AnOTA supported the suitability of its use for OTA detoxification in food and feed.

Inactive enzyme homologs, or pseudoenzymes, are proteins, found within most enzyme families, that are incapable of performing catalysis. Rather than catalysis, they are involved in protein-protein interactions, sometimes regulating the activity of their active enzyme cousins, or scaffolding protein complexes. Pseudoenzymes found within metallopeptidase families likewise perform these functions. Pseudoenzymes within the M14 carboxypeptidase family interact with collagens within the extracellular space, while pseudopeptidase members of the M12 \"a disintegrin and metalloprotease\" (ADAM) family either discard their pseudopeptidase domains as unnecessary for their roles in sperm maturation or utilize surface loops to enable assembly of key complexes at neuronal synapses. Other metallopeptidase families contain pseudopeptidases involved in protein synthesis at the ribosome and protein import into organelles, sometimes using their pseudo-active sites for these interactions. Although the functions of these pseudopeptidases have been challenging to study, ongoing work is teasing out the secret lives of these proteins.

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 μg/L OTA was completely degraded by 1.0 μg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-β-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.

Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization.
RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components.
CONCLUSIONS: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Methotrexate is an essential drug in the treatment of childhood cancer that is not exempt from toxicities. Glucarpidase is a drug used to reduce the toxic concentration of plasma methotrexate in patients with delayed elimination or at risk of toxicity. We describe the characteristics of a cohort of pediatric patients that received glucarpidase and analyze its role in the treatment of toxicity induced by high doses of methotrexate (HDMTX). Retrospective observational study of all pediatric cancer patients who received glucarpidase between 2012 and 2022 at a single center. Fifteen patients were treated with a single dose of glucarpidase, eleven of them presented with acute lymphoblastic leukemia and received HDMTX at 5 g/m2 in 24-hour infusion. In eight patients, glucarpidase was administered during the first cycle of HDMTX. The indication in thirteen cases was acute renal failure with delayed elimination of plasma methotrexate. The median maximum creatinine was 1.22 mg/dl (0.68 2.01 mg/dl), with a median increase over its baseline level of 313%. All patients normalized renal function after glucarpidase administration, with a median methotrexate excretion time of 193 hours (42-312 hours). No grade ≥2 adverse events derived from carboxypeptidase administration. Eleven patients received new doses of HDMTX in subsequent cycles, without new episodes of serious toxicity. The use of glucarpidase is effective and safe in the treatment of acute renal failure and methotrexate elimination delay in pediatric cancer patients. Further HDMTX doses may be prescribed without additional toxicities.

In mammals, a limited number of proteases catalyze with acidic amino acids as substrates. At present, there are only three known proteases: CCPs, carboxypeptidase O (CPO), and aspartate acylase (ASPA). Human CPO is a digestive enzyme that prefers glutamate as a substrate. It locates to the apical membrane of intestinal epithelial and is glycosylated protein. CPO is difficult to purify as it is a GPI-anchored protein. To obtain purified CPO, a truncated form called hCPOΔC was designed, which removed the C-terminal sequence of hCPO and was followed by His tag. Firstly, the truncated variant hCPOΔC (residues 1-349) was cloned into pFastBac vector to construct bacmid. Then the verified bacmid was transfected into Sf9 cells for expression. After the protein was successfully expressed, cell medium was collected and incubated with Ni resins. The target protein was eluted by imidazole through affinity chromatography. A purification method of human CPO with deglutamylation activity was successfully established using insect cells expression system. Purified hCPOΔC could hydrolyze glutamate in polypeptides.

Listeria monocytogenes is an important human and animal pathogen able to cause an infection named listeriosis and is mainly transmitted through contaminated food. Among its virulence traits, the ability to form biofilms and to survive in harsh environments stand out and lead to the persistence of L. monocytogenes for long periods in food processing environments. Virulence and biofilm formation are phenotypes regulated by quorum sensing (QS) and, therefore, the control of L. monocytogenes through an anti-QS strategy is promising. This study aimed to identify, by in silico approaches, proteins secreted by lactic acid bacteria (LAB) potentially able to interfere with the agr QS system of L. monocytogenes. The genome mining of Lacticaseibacillus rhamnosus GG and Lactobacillus acidophilus NCFM revealed 151 predicted secreted proteins. Concomitantly, the three-dimensional (3D) structures of AgrB and AgrC proteins of L. monocytogenes were modeled and validated, and their active sites were predicted. Through protein-protein docking and molecular dynamic, Serine-type D-Ala-D-Ala carboxypeptidase and L,D-transpeptidase, potentially secreted by L. rhamnosus GG and L. acidophilus NCFM, respectively, were identified with high affinity to AgrB and AgrC proteins, respectively. By inhibiting the translocation of the cyclic autoinducer peptide (cyclic AIP) via AgrB, and its recognition in the active site of AgrC, these LAB proteins could disrupt L. monocytogenes communication by impairing the agr QS system. The application of the QS inhibitors predicted in this study can emerge as a promising strategy in controlling L. monocytogenes in food processing environment and as an adjunct to antibiotic therapy for the treatment of listeriosis.

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