Carboxypeptidase A Research Articles

Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents.

The chelation of protein-bound metal ions is typically thought to follow either a dissociative (D) or an associative (A) path. While the former mechanism involves the spontaneous dissociation of the metal from the protein prior to chelation, the latter route is characterized by the formation of an intermediate protein-metal-chelator ternary complex. Using the prototypical zinc protease carboxypeptidase A (CPA) and a variety of charged and neutral chelating agents, we demonstrate that inactivation of the enzyme (and likely other metalloproteins) proceeds through a split pathway with contributions from both D- and A-type mechanisms. In the case of charged chelators such as ethylenediaminetetraacetic acid (EDTA), the proportions of both paths could be tuned over a wide range through variation of the chelator concentration and the ionic strength, I (from ∼95% A type at low I values to ∼5% at high I values). By measuring the EDTA concentration and time dependence of CPA inactivation and fitting the obtained kinetic data to a modified time-dependent inhibition model, we obtained the rate constants for the A and D paths (kinact and koff, respectively) and the inhibition constant (KI) for the formation of the CPA-Zn2+-EDTA ternary complex, indicating that the decreased contribution of the A-type mechanism at high ionic strengths originates from a large (40-fold; at I = 0.5 M) increase in KI. This observation might be related to a triarginine motif in CPA that electrostatically steers negatively charged substrates into the active site and may therefore also guide carboxylate-bearing chelators toward the Zn2+ ion.

A peptides-based biosensor with target-triggered charge-switchable property for simple and sensitive detection of Granzyme B

Granzyme B (GrB) is a serine protease released by natural killer cells and cytotoxic T lymphocytes during immune responses, which not only plays a role in tumor diagnosis but also provides valuable guidance during tumor treatment. In this work, we have designed a charge-switching peptide to fabricate an electrochemical biosensor for quantitative analysis of GrB. Specifically, the designed zwitterionic peptide is in an electrically neutral state before activation, and a door lock structure (proline) is constructed by utilizing the selectivity of carboxypeptidase A (CPA) to the carboxy-terminus of the peptide chain. The door lock is opened when the target is present, allowing CPA to hydrolyze the peptide. At this time, the peptide will convert from neutral to positive, triggering the assembly of a positively charged peptide layer on the electrode surface, resulting in a signal change. Studies have shown that the biosensor has good analytical performance, with a detection range of 0.01 pM–8 pM and a detection limit as low as 3.5 fM. Moreover, the developed biosensor has been effectively applied to the analysis of clinical samples, demonstrating its ability to monitor tumor progression and treatment with clinical applications.

Enhancing the thermostability of carboxypeptidase A by a multiple computer-aided rational design based on amino acids preferences at β-turns

Carboxypeptidase A (CPA) with efficient hydrolysis ability has shown vital potential in food and biological fields. In addition, it is also the earliest discovered enzyme with Ochratoxin A (OTA) degradation activity. Thermostability plays an imperative role to catalyze the reactions at high temperatures in industry, but the poor thermostability of CPA restricts its industrial application. In order to improve the thermostability of CPA, flexible loops were predicted through molecular dynamics (MD) simulation. Based on the amino acid preferences at β-turns, three ΔΔG-based computational programs (Rosetta, FoldX and PoPMuSiC) were employed to screen three variants from plentiful candidates and MD simulations were then used to verify two potential variants with enhanced thermostability (R124K and S134P). Results showed that compared to the wild-type CPA, the variants S134P and R124K exhibited rise of 4.2 min and 7.4 min in half-life (t1/2) at 45 °C, 3 °C and 4.1 °C in the half inactivation temperature (T5010), in addition to increase by 1.9 °C and 1.2 °C in the melting temperature (Tm), respectively. The mechanism responsible for the enhanced thermostability was elucidated through the comprehensive analysis of molecular structure. This study shows that the thermostability of CPA can be improved by the multiple computer-aided rational design based on amino acid preferences at β-turns, broadening its industrial applicability of OTA degradation and providing a valuable strategy for the protein engineering of mycotoxin degrading enzymes.

The detoxification of ochratoxin A in wine and grape juice by different enzymes and evaluation of their effects on the quality

AbstractOchratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium genera. It is hepatotoxic, nephrotoxic and carcinogenic. The growing contamination of OTA in wine and grape juice is a great threat to human. In this study, different enzymes were screened for the detoxification of OTA in wine and grape juice. The results indicated that seven enzymes had OTA degradation capacity in the buffer, and the degradation rate of carboxypeptidase A (CPA), lipase, pancreatin and thermolysin were 100%, 100%, 75.72%, and 73.45%, respectively. In grape beverages, pancreatin and CPA showed the most promising result with OTA degradation of 53.24% and 61.56% at 37°C. The LC‐MS/MS analysis further indicated that OTA produced nontoxic OTα and phenylalanine under treatment with three enzymes (carboxypeptidase A, lipase, and pancreatin). Furthermore, pancreatin and CPA had a clarifying effect on wine and grape juice, but had a slight effect on the organic acids and flavor components of them. It suggested that pancreatin and CPA could be used for OTA degradation in grape beverages.

Effect of dietary protein and lipid levels on growth performances and digestive enzymes activities in grouper (Epinephelus coioides)

This study investigates the effect of dietary protein and lipid levels on growth and digestive enzymes activities in grouper (Epinephelus coioides). Furthermore, grouper juveniles (8.63±0.47 g) were separated into ten groups and cultivated in a 100-L tank. Five groups were fed in different protein levels of 35, 40, 45, 50, and 55% protein, while the remaining were fed in different lipid levels of 6, 8, 10, 12, and 14% lipid. All experimental diets were mixed with 1.0% Sauropus androgynus extract. The fishes were sampled for digestive enzyme activities at 1, 4, and 7 days. The results showed that fish receiving 55% protein affected the growth and increased the activities of protease, lipase, carboxypeptidase A (CPA), and carboxypeptidase B (CPB). Liver tissue included higher levels of lipase, as well as carboxypeptidase A and B, while digestive tract tissue contained measurable levels of protease. In conclusion, the administration of grouper juveniles with S. androgynus extract can affect growth performances and increase digestive enzyme activities.

Biodegradation of ochratoxin A by endophytic Trichoderma koningii strains

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus spp. and Penicillium spp. that causes a threat to food safety and human health. Fungal biodegradation might be a promising strategy for reducing the OTA contamination in the future. In this study, the ability of Trichoderma koningii strains to degrade OTA produced by Aspergillus niger T2 (MW513392.1) isolated from tomato seeds was investigated. Among T. koningii strains tested, three strains; AUMC11519, AUMC11520 and AUMC11521 completely eliminated OTA from the culture medium, while AUMC11522 strain eliminated only 41.82% of OTA. OTα-amide, 3-phenylpropionic acid, OTα and phenylalanine were assayed as degradation products by FTIR analysis and LC–MS/MS spectra. Carboxypeptidase A (CPA) was found responsible for OTA degradation when a metal ion chelator, EDTA, was added to cell free supernatants of the three effective strains. OTA detoxification by T. koningii could present new prospective strategies for a possible application in food commodities intoxicated with ochratoxin.

Anabaenopeptins from Nostoc edaphicum CCNP1411.

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.

Serum Carboxypeptidase Activity and Genotype-Stratified CA19-9 to Detect Early-Stage Pancreatic Cancer

Serum diagnostic markers of early-stage pancreatic ductal adenocarcinoma (PDAC) are needed, especially for stage I disease. As tumors grow and cause pancreatic atrophy, markers derived from pancreatic parenchyma such as serum carboxypeptidase A (CPA) activity lose diagnostic performance. We evaluated, with CA19-9, serum CPA as a marker of early pancreatic cancer. Serum CPA activity levels were measured in 345 controls undergoing pancreatic surveillance, divided into 2 sets, set 1 being used to establish a reference range. Variants within the CPA1 locus were sought for their association with pancreatic CPA1 expression to determine if such variants associated with serum CPA levels. A total of 190 patients with resectable PDAC were evaluated. Among controls, those having 1 or more minor alleles of CPA1 variants rs6955723 or rs2284682 had significantly higher serum CPA levels than did those without (P= .001). None of the PDAC cases with pancreatic atrophy had an elevated CPA. Among 122 PDAC cases without atrophy, defining serum CPA diagnostic cutoffs by a subject's CPA1 variants yielded a diagnostic sensitivity of 18% at 99% specificity (95% confidence interval [CI], 11.7-26) (vs 11.1% sensitivity using a uniform diagnostic cutoff); combining CPA with variant-stratified CA19-9 yielded a sensitivity of 68.0% (95% CI, 59.0-76.2) vs 63.1% (95% CI, 53.9- 71.7) for CA19-9 alone; and among stage I PDAC cases, diagnostic sensitivity was 51.9% (95% CI, 31.9-71.3) vs 37.0% (95% CI, 19.4-57.6) for CA19-9 alone. In the validation control set, the variant-stratified diagnostic cutoff yielded a specificity of 98.2%. Serum CPA activity has diagnostic utility before the emergence of pancreatic atrophy as a marker of localized PDAC, including stage I disease.

Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A.

Ochratoxin A (OTA) is a toxic secondary metabolite produced mainly by Penicillium spp. and Aspergillus spp. and commonly found in foodstuffs and feedstuffs. Carboxypeptidase A (CPA) can hydrolyze OTA into the non-toxic product ochratoxin α, with great potential to realize industrialized production and detoxify OTA in contaminated foods and feeds. This study constructed a P. pastoris expression vector of mature CPA (M-CPA) without propeptide and signal peptide. The results showed that the degradation rate of OTA by M-CPA was up to 93.36%. Its optimum pH was 8, the optimum temperature was 40 °C, the value of Km was 0.126 mmol/L, and the maximum reaction rate was 0.0219 mol/min. Compared with commercial CPA (S-CPA), the recombinant M-CPA had an improve stability, for which its optimum temperature increased by 10 °C and stability at a wide range pH, especially at pH 3–4 and pH 11. M-CPA could effectively degrade OTA in red wine. M-CPA has the potential for industrial applications, such as can be used as a detoxification additive for foods and feeds.

Carboxypeptidase A immobilization with zeolitic imidazolate framework for enhancement of ochratoxin A degradation ability

ABSTRACT Ochratoxin A (OTA) is a highly toxic potential contaminant of most agricultural products, and OTA is difficult to remove in general food production processes. In this study, we immobilized polyvinyl pyrrolidone-coated carboxypeptidase A (CPA) into zeolitic imidazolate framework material to improve the ability for OTA degradation. In this method, carboxypeptidase A was dispersed well in the material and maintained its stability and ability to degrade OTA, under complex matrix conditions with better repeated use performance than free carboxypeptidase A. The substrate conversion rate of CPA increased 3 times after immobilization. After repeated use of 10 cycles, the composite enzyme still retained 71.37% of its initial activity. The degradation rate of OTA was 30.69% higher than that of free CPA. This method may play an important role in the degradation of mycotoxins.

Comparative studies on the interaction of spermidine with carboxypeptidase A using multispectroscopic and docking methods

Carboxypeptidase A (CPA) (EC 3.4.17.1) is one of the main members of the M14 family that release one amino acid from the C-terminal region of the polypeptides at each time. The purpose of the present study was to study the effect of spermidine (NH2(CH2)3NH(CH2)4NH2) on the conformation, thermal stability, and activity of native CPA from bovine pancreas, by employing ultraviolet–visible (UV–Vis) spectroscopy, intrinsic fluorescence, thermal stability, circular dichroism (CD), kinetic techniques and molecular docking. It was found that the decrease in the CPA, UV–Vis absorption could be due to the formation of the CPA–spermidine complexes. The results of fluorescence spectroscopic measurements at the temperatures of 308 and 318 K also revealed that spermidine had the capability to quench the intrinsic fluorescence of CPA with the static mode. Further, the thermodynamic parameters, (Gibbs free-energy, enthalpy and entropy changes) showed that the binding process of spermidine to CPA was spontaneous and the main force in stabilizing the complex was the van der Waals and hydrogen interactions, along with the molecular docking results. In addition, CD spectra and fluorescence results revealed that spermidine had a partial effect on the CPA structure, leading to some changes in its secondary structure. The Tm studies of the CPA–spermidine complex also indicated that the Tm values were enhanced with increasing the spermidine concentration. Kinetic studies further showed that by spermidine binding, the Vmax value and activity of the enzyme were increased.

Mast cell peptidases (carboxypeptidase A and chymase)-mediated hydrolysis of human angiotensin-(1–12) substrate

Angiotensin processing peptidases (carboxypeptidase A (CPA) and chymase) are stored in cardiac mast cell (MC) secretory granules in large quantity and are co-released into the extracellular environment after activation/degranulation. In the human heart, chymase is primarily responsible for angiotensin II (Ang II) generation from the alternate substrate angiotensin-(1–12) (Ang-(1–12)). We investigated the individual and combined hydrolytic specificity of CPA and chymase enzymes (1:1 and 1:⅓ ratio) in the processing of the human Ang-(1–12) (hAng-(1–12)) substrate. To determine the Km and Vmax, the CPA and recombinant human chymase (rhChymase) enzymes were incubated with increasing concentrations of hAng-(1–12) substrate (0–300 μM). We found that CPA alone sequentially metabolized hAng-(1–12) substrate into angiotensin-(1–9) (Ang-(1–9), 53%), Ang II (22%) and angiotensin-(1–7) (Ang-(1–7), 11%) during a 15 min incubation. In the presence of rhChymase alone, 125I-hAng-(1–12) was directly metabolized into Ang II (89%) and no further hydrolysis of Ang II was detected. In the presence of both CPA + rhChymase enzymes (1:1 or 1:⅓ ratio), the amount of Ang II formation from 125I-hAng-(1–12) within a 5 min incubation period were 68% or 65%, respectively. In the presence of both (CPA + rhChymase), small amounts of Ang-(1–9) and Ang-(1–7) were generated from 125I-hAng-(1–12). The Km and Vmax values were 150 ± 5 μM and 384 ± 23 nM/min/mg of CPA and 40 ± 9 μM and 116 ± 20 nM/min/mg of rhChymase. The catalytic efficiency (Vmax/Km ratio) was higher for rhChymase/hAng-(1–12) compared to CPA/hAng-(1–12). Compared to CPA, chymase has a much higher affinity to hydrolyze the hAng-(1–12) substrate directly into Ang II. In addition, Ang II and Ang-(1–7) are the end products of chymase and CPA, respectively. Overall, our findings suggest that the Ang II generation from hAng-(1–12) is primarily mediated by chymase rather than CPA.

Mast Cell Peptidases (Carboxypeptidase A and Chymase)‐Mediated Hydrolysis of Human Angiotensin‐(1‐12)

IntroductionAngiotensin processing peptidases [carboxypeptidase A (CPA) and chymase] are stored in cardiac mast cell secretory granules and are co‐released into the extracellular environment after activation/degranulation. In the human heart, we showed that chymase is primarily responsible for angiotensin II (Ang II) generation from the alternate substrate angiotensin‐(1‐12) [Ang‐(1‐12)], which is present in human heart. In this study, we investigated whether there are any synergistic effects of CPA and chymase in the processing of Ang‐(1‐12).MethodRadiolabeled human Ang‐(1‐12) [125I‐Ang‐(1‐12)] substrate was incubated with CPA alone (0.325 μg/mL), human recombinant chymase (hrChymase) (0.325 μg/mL) or a combination of CPA + hrChymase (1:1 or 1: ⅓ ratio) in 50 mM Tris‐HCl buffer solution containing 150 mM NaCl (pH 8.0) for 0–15 min at 37 °C. The reaction was stopped by adding an equal volume of 1% phosphoric acid and injected on a C18 column. Eluted 125I‐Ang‐(1‐12) metabolic products were quantified by HPLC connected to in‐line flow‐through gamma detector. To determine the Km and Vmax, CPA and hrChymase were incubated with increasing concentrations (0–300 μM) of human Ang‐(1‐12) substrate for 20 min at 37 °C. The Km and Vmax were calculated using the Michaelis‐Menten equation.ResultsWe found that after 15 min of incubation, CPA sequentially metabolized Ang‐(1‐12) substrate into angiotensin‐(1‐9) [Ang‐(1‐9), 53% (major product)], Ang II (22%) and angiotensin‐(1‐7) [Ang‐(1‐7), 11%]. No further hydrolysis of Ang‐(1‐7) was detected during CPA incubation. In the presence of hrChymase alone, 125I‐Ang‐(1‐12) was directly metabolized into Ang II (89%) and no further hydrolysis of Ang II was detected. In the presence of both CPA + hrChymase (1:1 ratio), the amount of Ang II formation was 68% from 125I‐Ang‐(1‐12) within a 5 min incubation period. The formation of Ang‐(1‐9) and Ang‐(1‐7) were only 11% and 10%, respectively. When CPA + hrChymase were added in 1: ⅓ ratio for 5 min, the Ang II, Ang‐(1‐9) and Ang‐(1‐7) formations were 65%, 27%, 6%, respectively. The Km and Vmax values were 150 ± 5 μM and 384 ± 23 nM/min/mg of CPA and 40 ± 9 μM and 116 ± 20 nM/min/mg of hrChymase.ConclusionsThese findings suggest that compared to CPA, chymase shows a much higher affinity to metabolize Ang‐(1‐12) into Ang II. In addition, Ang II and Ang‐(1‐7) are the end products of chymase and CPA, respectively. Overall, our findings suggest that chymase, rather than CPA, is primarily responsible for Ang II generation from Ang‐(1‐12).Support or Funding InformationThis work was supported by grant from the National Heart, Lung and Blood Institute of the National Institutes of Health (P01 HL‐051952).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nuclear receptor hormone receptor 39 is required for locust moulting by regulating the chitinase and carboxypeptidase genes.

The nuclear receptor-mediated 20-hydroxyecdysone (20E) signalling pathway plays crucial roles in insects by initiating and regulating moulting and metamorphosis. In the present study, we identified and characterized a cDNA encoding a putative nuclear receptor protein (Locusta migratoria hormone receptor 39, LmHR39) based on L.migratoria transcriptomics data. Reverse-transcription quantitative PCR (RT-qPCR) revealed that LmHR39 shows low-level expression in the early days of fifth-instar nymphs, and peak expression occurs on day 5, which is followed by a decrease before ecdysis. LmHR39 transcription could be induced by 20E in vivo and was significantly suppressed by knocking down the expression of the L.migratoria ecdysone receptor gene and early-late gene LmHR3. After RNA interference of LmHR39 with double-stranded RNA (dsRNA), 85% of the insects showed abnormal morphology, with curly wings after moulting and delayed eclosion time. Haematoxylin and eosin staining indicated that apolysis of the integument and wing pad cuticle in the dsLmHR39-treated insects was delayed compared to that in the dsRNA for green fluorescent protein-injected control. Furthermore, RNA-sequencing and RT-qPCR analysis showed the expression level of carboxypeptidase genes (Carboxypeptidase A (CPA) and Carboxypeptidase M (CPM)) and chitin degrading genes (LmChitinase5 (LmCHT5) and LmChitinase10 (LmCHT10)) dramatically declined in the dsLmHR39-treated insects, implying that the LmHR39-mediated 20E signalling pathway is involved in the regulation of carboxypeptidase genes (CPA and CPM) and chitinase genes (LmCHT5 and LmCHT10), and participated in apolysis of the integument and wing pads during locust moulting.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

Immobilization of Carboxypeptidase A into Modified Chitosan Matrixes by Covalent Attachment.

Carboxypeptidase A (CPA) is a metalloexopeptidase that catalyzes the hydrolysis of the peptide bonds that are adjacent to the C-terminal end of a polypeptide chain. The enzyme preferentially cleaves over C-terminal L-amino acids with aromatic or branched side chains. This is of main importance for food industry because it can be employed for manufacturing functional foods from different protein sources with reduced hydrophobic amino acid content for patients with deficiencies in the absorption or digestion of the corresponding amino acids. In that way, strategies for effective multipoint covalent immobilization of CPA metalloenzyme on chitosan beads have been developed. The study of the ability to produce several chemical modifications on chitosan molecules before, during and after its coagulation to form carrier beads lead in a protective effect of the polymer matrix. The chemical modification of chitosan through the use of an N-alkylation strategy produced the best derivatives. N-alkyl chitosan derivative beads with D-fructose presented values of 0.86 for immobilization yield, 314.6IUg-1 bead for initial activity of biocatalyst and were 5675.64-fold more stable than the free enzyme at 55°C. Results have shown that these derivatives would present a potential technological application in hydrolytic processes due to both their physical properties, such as low swelling capacity, reduced metal chelation ability and bulk mesoporosity, and increased operational stability when compared with soluble enzyme.

English

Carboxypeptidase A (CPAs) are a well-studied group of zinc-containing exopeptidases that facilitate the breakdown of proteins and peptides during metabolism. Carboxypeptidase A is typically produced in mammalian pancreatic, brain and other tissues. A new gene encoding carboxypeptidase A in the prokaryote Bacillus pumilus was amplified by polymerase chain reaction (PCR), ligated into the shuttle vector pMA5, and cloned in a GRAS bacteria-Bacillus subtilis 168 host. This gene sequence contained a 1621 bp open reading frame that encodes a protein of 540 amino acids. The optimum pH and temperature for enzyme activity were 7.5 and 50°C, respectively. The enzyme was quite stable at neutral pH and maintained about 65% activity following a 24 h incubation at 40°C. The Km of this CPA was 0.1 mM, much higher than in mammalian species. Glycerol, ammonium sulfate, and sodium citrate improved enzyme activity under optimal culture condition. The carboxypeptidase activity in recombinant B. subtilis 168 reached a maximum of 179 U ml-1 in a 5 L fermentator when cultured on improved medium. The over expression of carboxypeptidase A in Bacillus subtilis has commercial applications. Key words: Bacillus pumilus, Bacillus subtilis 168, over-expression, orthogonal arrays, carboxypeptidase A, metallocarboxypeptidase.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. DisclosuresNo relevant conflicts of interest to declare.

Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases.

We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.

How my Experiences at the MRC-LMB led to the Structure of the Ribosome

Max Perutz's Dunham Lectures at Harvard in 1963, in which he showed the first atomic structure of a protein (myoglobin) that anyone in the room had ever seen, led to my working on the structure of carboxypeptidase A (CPA) (in the Lipscomb lab) before going to the MRC LMB in Cambridge. "Rapid" data collection on CPA in the mid-1960s was 5,000 reflections in a week, and now obtaining 2.7 Å resolution data on crystals with two 70S ribosomes in the asymmetric unit takes 5 minutes. Importantly, the LMB promoted creative and novel science because of its cooperative, interactive atmosphere where everyone interacted in the hall or over coffee, lunch or tea. This influenced how I have carried out science over the subsequent years. In the canteen, Crick, Brenner, Perutz, etc., would be interacting and talking with postdocs and students about asking important questions and solving scientific problems. It was a great place to learn, develop and use the most advanced methods in protein crystallography and apply them to explore the most interesting and significant questions in molecular biology. The importance of integrating structure and function in our research goals was made clear. My interactions in the Cambridge LMB led me to pursue the structural basis for understanding Crick's Central Dogma of Molecular Biology – DNA makes DNA makes RNA makes proteins. This resulted in our ultimately determining the structures of the ribosome and its various complexes.