Tandem Targeting and Dual Aggregation of an AIEgen for Enhanced Near-Infrared Fluorescence Imaging of Tumors.

Near-infrared (NIR) aggregation-induced emission luminogens (AIEgens) are excellent probes for tumor imaging, but there still is space to improve their imaging specificity and sensitivity. In this work, a strategy of tandem targeting and dual aggregation of an AIEgen is proposed to achieve these two purposes. An AIEgen, ß-tBu-Ala-Cys(StBu)-Lys(Biotin)-Pra(QMT)-CBT (Ala-Biotin-QMT), is designed to tandem target the biotin receptor and leucine aminopeptidase of a cancer cell and thereafter undergo CBT-Cys click reaction-mediated dual aggregations in the cell. Experimental results show that Ala-Biotin-QMT renders 4.8-fold and 7.9-fold higher NIR fluorescence signals over those in the "biotin + LAP inhibitor"-treated control groups in living HepG2 cells and HepG2 tumor-bearing mice, respectively. We anticipate that Ala-Biotin-QMT, which has the tandem targeting and dual aggregation property to simultaneously achieve enhanced tumor enrichment and fluorescence onset, could be applied for accurate cancer diagnosis in the clinic in the future.

Dual Enzyme-Locked Activation Reporter for Accurate Liver Cancer Surveillance.

Activatable probes with a higher signal-to-background ratio and accuracy are essential for monitoring liver cancer as well as intraoperative fluorescence navigation. However, the presence of only one biomarker is usually not sufficient to meet the high requirement of a signal-to-background ratio in cancer surveillance, leading to the risk of misdiagnosis. In this work, a dual-locked activation response probe, Si-NTR-LAP, for nitroreductase and leucine aminopeptidase was reported. This dual-locked probe provides better tumor recognition and a higher signal-to-noise ratio than that of single-locked probes (Si-LAP and Si-NTR). In both the subcutaneous tumor model and the more complex orthotopic hepatocellular carcinoma model, the probe was able to identify tumor tissue with high specificity and accurately differentiate the boundaries between tumor tissue and normal tissue. Therefore, the dual-locked probe may provide a new and practical strategy for applying to real patient tumor tissue samples.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Unveiling the Catalytic Mechanism of a Processive Metalloaminopeptidase.

Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PaPepA: a leucine aminopeptidase from Pseudomonas aeruginosa. Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PaPepA revealed that PaPepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PaPepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.

The aminopeptidase LAP3 suppression accelerates myogenic differentiation via the AKT-TFE3 pathway in C2C12 myoblasts.

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

Leucine Aminopeptidase-Mediated Multifunctional Molecular Imaging Tool for Diagnosis, Drug Evaluation, and Surgical Guidance of Liver-Related Diseases.

Traditional molecular imaging tools used for detecting liver diseases own several drawbacks, such as poor optical performance and limited applicability. Monitoring the concentration of leucine aminopeptidase (LAP), which is closely related to liver diseases such as liver cancer and liver injury, and analyzing it in diagnosis, drug evaluation, and surgical treatment is still a challenging task. Herein, we construct an intramolecular charge-transfer mechanism-based, ultrasensitive, near-infrared fluorescent probe (LAN-lap) for dynamic monitoring of LAP fluctuations in living systems. LAN-lap, with high specificity, stability, sensitivity, and water solubility, can achieve in vitro monitoring of LAP through both fluorescence and colorimetric methods. Moreover, LAN-lap can successfully be used for the localization imaging of endogenous LAP, confirming the upregulation of LAP expression in liver cancer and liver injury cells. In addition, LAN-lap can realize the imaging of liver tumors in living organisms. Meanwhile, it can intuitively present the degree of drug-induced liver injury, achieving semi-quantitative imaging evaluation of the hepatotoxicity of two drugs. Furthermore, LAN-lap can track liver cancer tumors in mice with peritoneal metastasis and can assist in fluorescence-guided surgical resection of liver cancer tumors. This multifunctional LAN-lap probe could play an important role in facilitating simultaneous diagnoses, imaging, and synergistic surgical navigation to achieve better point-of-care therapeutic efficacy.

Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Synthetic and biological surfactant effects on freshwater biofilm community composition and metabolic activity.

Surfactants are used to control microbial biofilms in industrial and medical settings. Their known toxicity on aquatic biota, and their longevity in the environment, has encouraged research on biodegradable alternatives such as rhamnolipids. While previous research has investigated the effects of biological surfactants on single species biofilms, there remains a lack of information regarding the effects of synthetic and biological surfactants in freshwater ecosystems. We conducted a mesocosm experiment to test how the surfactant sodium dodecyl sulfate (SDS) and the biological surfactant rhamnolipid altered community composition and metabolic activity of freshwater biofilms. Biofilms were cultured in the flumes using lake water from Lake Lunz in Austria, under high (300 ppm) and low (150 ppm) concentrations of either surfactant over a four-week period. Our results show that both surfactants significantly affected microbial diversity. Up to 36% of microbial operational taxonomic units were lost after surfactant exposure. Rhamnolipid exposure also increased the production of the extracellular enzymes, leucine aminopeptidase, and glucosidase, while SDS exposure reduced leucine aminopeptidase and glucosidase. This study demonstrates that exposure of freshwater biofilms to chemical and biological surfactants caused a reduction of microbial diversity and changes in biofilm metabolism, exemplified by shifts in extracellular enzyme activities. KEY POINTS: • Microbial biofilm diversity decreased significantly after surfactant exposure. • Exposure to either surfactant altered extracellular enzyme activity. • Overall metabolic activity was not altered, suggesting functional redundancy.

Sperm-Leucylaminopeptidases are required for male fertility as structural components of mitochondrial paracrystalline material in Drosophila melanogaster sperm.

Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of S-Lap proteins we show the characterization of classical mutants and RNAi lines affecting of the S-Lap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the S-Lap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that S-Lap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that S-Lap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.

The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase.

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling.

BACKGROUND: Durian (Durio zibethinus L.) is a highly popular fruit in Thailand and several other Southeast Asian countries. It is abundant in essential nutrients and sulphur-containing compounds such as glutathione (GSH) and γ-glutamylcysteine (γ-EC). Cysteinylglycine (Cys-Gly) is produced by GSH catabolism and occurs in durian fruit pulp. Cysteine (Cys) is a precursor of sulphur-containing volatiles generated during fruit ripening. The aforementioned substances contribute to the strong odour and flavour of the ripe fruit. However, the genes encoding plant Cys-Gly dipeptidases are unknown. The aim of this study was to measure leucylaminopeptidase (LAP) activity in durian fruit pulp. RESULTS: We identified DzLAP1 and DzLAP2, which the former was highly expressed in the fruit pulp. DzLAP1 was expressed at various ripening stages and in response to ethephon/1-MCP treatment. Hence, DzLAP1 is active at the early stages of fruit ripening. DzLAP1 is a metalloenzyme ~ 63 kDa in size. It is activated by Mg2+ or Mn2+ and, like other LAPs, its optimal alkaline pH is 9.5. Kinetic studies revealed that DzLAP1 has Km = 1.62 mM for its preferred substrate Cys-Gly. DzLAP1-GFP was localised to the cytosol and targeted the plastids. In planta Cys-Gly hydrolysis was confirmed for Nicotiana benthamiana leaves co-infiltrated with Cys-Gly and expressing DzLAP1. CONCLUSIONS: DzLAP1 has Cys-Gly dipeptidase activity in the γ-glutamyl cycle. The present study revealed that the LAPs account for the high sulphur-containing compound levels identified in fully ripened durian fruit pulp.

Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance.

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

Digestive and immune functions in the intestine of wild Ballan wrasse (Labrus bergylta).

This study was carried out to profile key characteristics of intestinal functions and health in wild-caught Ballan wrasse. To describe functional variation along the intestine, samples were collected from four intestinal segments, named from the proximal to the distal segment: IN1, IN2, IN3 and IN4. The sections showed quite similar structure, i.e. regarding mucosal fold height and branching, lamina propria and submucosal width and cellular composition and thickness of the muscle layers. Leucine aminopeptidase and maltase capacity decreased from IN1 to IN4, suggesting a predominant role of IN1 in digestion. Gene expression levels of vitamin C transporter (slc23a1) and fatty acid transporters (cd36 and fabp2) were higher in IN1 than in IN4, indicating a more important role of the proximal intestine regarding transport of vitamins and fatty acids. Higher expression of the gene coding for IgM heavy chain constant region (ighm) was found in IN4 than in IN1, suggesting an important immune function of the distal intestine. Other immune related genes il1b, il6, cd40, showed similar expression in the proximal and the distal part of the intestine. Parasite infection, especially the myxozoan parasite Enteromyxum leei, coincided with infiltration of lymphocytic and eosinophilic granular cells in the submucosa and lamina propria. The present study established reference information necessary for interpretation of results of studies of intestinal functions and health in cultured Ballan wrasse.

Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum.

The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8(+) T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) major histocompatibility complex class I molecule. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8(+) T cell populations was impaired in T. gondii-infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.

High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis.

Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30-80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65-75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

Leucine aminopeptidase 3 promotes migration and invasion of breast cancer cells through upregulation of fascin and matrix metalloproteinases-2/9 expression.

Overexpression of leucine aminopeptidase 3 (LAP3) is involved in proliferation, migration, and invasion of several tumor cells and plays a crucial role in tumor metastasis. However, the related mechanism remains unknown. In this study, we used MDA-MB-231 and MCF7 breast cancer cell lines to explore the role of LAP3 in the regulation of cancer cell migration and invasion by employing the natural LAP3 inhibitor bestatin and a lentivirus vector that overexpresses or knocks down LAP3. Bestatin inhibited tumor cell migration and invasion in a dose-dependent manner. Western blot assay showed that bestatin and knockdown of LAP3 upregulated phosphorylation of Hsp27 and downregulated expression of fascin. Phosphorylation of Akt and expression of matrix metalloproteinase-2/9 can also be downregulated. LAP3 overexpression showed the opposite results. Immunohistochemistry analysis was conducted to detect expression levels of LAP3 in breast cancer tissues. High LAP3 expression was correlated with the grade of malignancy. Findings of this study uncovered the molecular mechanism of LAP3 on breast cancer metastasis and indicated that LAP3 may act as a potential antimetastasis therapeutic target.

De Novo Design of Chemical Stability Near-Infrared Molecular Probes for High-Fidelity Hepatotoxicity Evaluation In Vivo.

Near-infrared (NIR) fluorescence imaging technique is garnering increasing research attention due to various advantages. However, most NIR fluorescent probes still suffer from a false signals problem owing to their instability in real application. Especially in a pathological environment, many NIR probes can be easily destroyed due to the excessive generation of highly reactive species and causing a distorted false signal. Herein, we proposed an approach for developing a new stable NIR dye platform with an optically tunable group to eliminate false signals using the combination of dyes screening and rational design strategy. The conception is validated by the construction of two high-fidelity NIR fluorescent probes (NIR-LAP and NIR-ONOO-) sensing leucine aminopeptidase (LAP) and peroxynitrite (ONOO-), the markers of hepatotoxicity. These probes (NIR-LAP and NIR-ONOO-) were demonstrated to sensitively and accurately monitor LAP and ONOO- (detection limit: 80 mU/L for LAP and 90 nM for ONOO-), thereby allowing one to precisely evaluate drug-induced hepatotoxicity. In addition, based on the fluctuation of LAP, the therapeutic efficacy of six hepatoprotective medicines for acetaminophen-induced hepatotoxicity was analyzed in vivo. We anticipate the high-fidelity NIR dye platform with an optically tunable group could provide a convenient and efficient tool for the development of future probes applied in the pathological environment.

A leucine aminopeptidase is involved in kinetoplast DNA segregation in Trypanosoma brucei.

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks.

A near-infrared fluorescent probe for monitoring leucine aminopeptidase in living cells.

Leucine aminopeptidase (LAP), one of the important cancer-related biomarkers, is significantly over-expressed in many malignant tumor cells. Developing an effective fluorescent probe for high-specificity and in situ trapping of endogenous LAP in living samples is still challenging. In this project, we report a water-soluble near-infrared (NIR) fluorescent probe (CHMC-M-Leu) for specific monitoring of LAP in vitro and in vivo. The novel fluorescent probe (CHMC-M-Leu) contains a NIR-emitting fluorophore (CHMC-M) as the reporter and l-leucine as the enzyme-active trigger moiety which are linked together by a p-aminobenzyl alcohol (PABA) section. Upon exposure to LAP, the fluorescence at 625 nm gets impressively enhanced, which belongs to the near-infrared region and is beneficial for imaging in vivo. Furthermore, the novel fluorescent probe exhibits fast response and highly chemoselective detection of LAP in various bio-related species. In addition, CHMC-M-Leu shows favourable cellular uptake and was successfully used to monitor endogenous LAP in living cells.