Cell-based analysis of structure-function activity of threonine aspartase 1.

Area of Science:

• Biochemistry and Molecular Biology
• Protease Function and Regulation
• Cellular Assays and Biosensors

Background:

• Taspase1 is a threonine protease involved in intracellular substrate cleavage, crucial for development and potentially disease.
• The complete set of Taspase1 targets and its precise cleavage requirements remain largely unknown.
• The absence of cellular assays hinders the study of Taspase1's biological and pathological relevance.

Purpose of the Study:

• To develop and validate cell-based translocation biosensor assays for probing Taspase1 trans-cleavage in vivo.
• To identify the sequence and spatial requirements for efficient Taspase1 substrate processing.
• To determine the human Taspase1 degradome and identify novel bona fide targets.

Main Methods:

• Development of modular, cell-based translocation biosensor assays with varying Taspase1 cleavage sites localized to the cytoplasm.
• Utilizing scanning mutagenesis in conjunction with the biosensor assay to define Taspase1 processing requirements.
• Genome-wide bioinformatic analysis to identify potential Taspase1 targets based on the refined consensus sequence.

Main Results:

• Successfully established efficient cell-based biosensor assays that detect Taspase1 activity via proteolytic cleavage and nuclear translocation.
• Identified an improved Taspase1 consensus recognition sequence: Q(3)(F/I/L/V)(2)D(1)↓G(1)'X(2)'D(3)'D(4)'.
• Discovered 27 putative Taspase1 targets, including cytoplasmic and nuclear proteins like USF2 and NXF2/5, with verified cleavage.

Conclusions:

• The developed biosensor assays provide a powerful tool for studying Taspase1 activity in cellular contexts.
• The refined consensus sequence enables the first genome-wide identification of the human Taspase1 degradome.
• These findings offer novel mechanistic insights into Taspase1 function and its potential role in disease.