Profiling Cystathionine β/γ-Lyase in Complex Biosamples Using Novel Activatable Fluorogens.

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Cystathionine lyase is crucial in transsulfuration pathways, impacting numerous physiological and pathophysiological processes.
• Current methods for monitoring cystathionine lyase activity in biological samples are limited, leading to unreliable results.
• The hydrogen sulfide/cystathionine lyase system's role in disease requires precise molecular monitoring tools.

Purpose of the Study:

• To design and develop novel activatable fluorescent probes for direct monitoring of cystathionine lyase activity.
• To create probes based on the naphthylamide scaffold with specific recognition moieties and self-immolative linkers.
• To establish reliable methods for quantifying cystathionine lyase (CBL and CSE) levels in complex biological systems.

Main Methods:

• Development of naphthylamide-based fluorescent probes (CBLP and CSEP) incorporating cysteine/homocysteine recognition and a carbamate ethyl sulfide linker.
• In vitro assessment of probe selectivity and sensitivity for cystathionine beta-lyase (CBL) and cystathionine gamma-lyase (CSE).
• Application of probes in various biological models, including plant roots, cell lines, zebrafish, and septic rat liver tissue.

Main Results:

• CBLP demonstrated high selectivity and sensitivity for semiquantifying CBL levels in Arabidopsis thaliana roots and mutants.
• CSEP successfully detected CSE levels in HCC-LM3 cells, zebrafish, and upregulated CSE in septic rat liver tissue.
• Probe performance was validated using Western blotting and immunohistochemical analysis, confirming their efficacy.

Conclusions:

• The developed fluorescent probes provide an effective strategy for reporting cystathionine lyase activity.
• This approach facilitates the profiling of cystathionine lyase in diverse biological processes.
• The study paves the way for accurate and efficient direct estimation of cystathionine lyase activity in biological research and diagnostics.