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A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

ABSTRACT: Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to increase its water solubility and activation kinetics by addition of a long PEG chain (5 kDa) at the C-terminus. A BODIPY fluorophore allowed detection of Cathepsin D across a wide pH range, important as the protease is active both at the low pH found in lysosomes and also in higher pH phagolysosomes, and in the cytosol. The probe was successfully used to detect Cathepsin D activity in macrophages challenged by exposure to bacteria.

SUBMITTER: Rodriguez-Rios M

PROVIDER: S-EPMC10622513 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

Rodriguez-Rios Maria M McHugh Brian J BJ Liang Zhengqi Z Megia-Fernandez Alicia A Lilienkampf Annamaria A Dockrell David D Bradley Mark M

Communications chemistry 20231102 1

Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to ...[more]

PMID: 37919467

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Project description:A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

Project description:Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate (SQ-3 PC) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes SQ-1, SQ-2, and SQ-3 along with a dye-peptide conjugate SQ-3-PC as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing SQ-3 and SQ-3 PC respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing SQ-3 and SQ-3 PC, respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe SQ-3 PC demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of SQ-3 PC led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that SQ-3 PC works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.

Dataset Information

A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

Publications

A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

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