Tau protease compositions and methods of use
Inventors
Moe, James G. • Davidowitz, Eliot J. • Lopez, Patricia
Assignees
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Publication Number
US-12060591-B2
Publication Date
2024-08-13
Expiration Date
Abstract
Disclosed are mammalian tau proteases, as well as proteolytically-active fragments, variants, and mutants thereof. Also disclosed are polynucleotides and recombinant expression vectors that encode these polypeptides, as well as methods for producing such proteins in selected recombinant host cells, and for using the compositions in a variety of diagnostic and analytical assays.
Core Innovation
The invention relates to mammalian tau proteases and tau protease variant peptides or polypeptides. Tau forms disulfide-mediated tau oligomers that become autoproteolytically active serine proteases, including Tau 4R2N species such as monomer, dimer, and trimer.
The document identifies principal autocatalytic cut sites in tau, including a cut site at Lys340-Ser341, and links these cut sites to the formation of active serine protease states. It also describes truncated tau proteases, including TP-210 (131-340) and TP-99 (241-340), that retain protease activity, supporting that tau functions as an active serine protease in defined fragments.
Protease activity is documented for tau cleavage of other disease-relevant substrates, including tubulin, APP, alpha-endorphin, and alpha-endorphin. The serine-protease class is supported by inhibitor profiling and active-site labeling using fluorophosphonate (FP) labeling and FP-TAMRA, in combination with serine protease inhibitors such as AEBSF/PMSF and chymostatin.
The document further describes FRET-based protease activity detection using tau-cleavable fluorescent peptide substrates, and it discusses nucleic-acid and immunodetection tools for tau protease-specific contexts, including PCR primers/probes and antibody/immunodetection reagents for tau protease-specific epitopic core regions. The overall system is presented as enabling protease activity detection and analytical or diagnostic use related to tau protease activity.
Claims Coverage
The independent claims cover tau protease variant peptides or polypeptides defined by substitutions at specified residue positions of a starting mammalian tau protease sequence, together with bacterial host cell expression, and enhanced serine protease activity compared to the original, un-substituted tau protease. Dependent claims further specify permissible substitution types and impose activity threshold constraints relative to a full-length Tau 4R2N trimer protease.
Tau protease variant substitutions at specified residue positions
A tau protease variant peptide or polypeptide comprising an amino acid sequence of a starting mammalian tau protease substituted at at least one amino acid position corresponding to one of the listed residue numbers of SEQ ID NO:6, wherein the substitution confers enhanced serine protease activity compared to the original, un-substituted tau protease peptide or polypeptide.
Enhanced serine protease activity when expressed in a bacterial host cell
The tau protease variant peptide or polypeptide is expressed in a bacterial host cell, and the enhanced serine protease activity is conferred by the specified substitutions.
Permissible amino-acid change types for the substitutions
The substitutions are limited to particular listed amino-acid change types for the specified positions, including singly or any combination of the listed substitution options.
Serine protease activity at least 85% of full-length Tau 4R2N trimer protease
The tau protease variant peptide or polypeptide has serine protease activity at least 85% of the serine protease activity of the full-length Tau 4R2N trimer protease.
Serine protease activity at least 95% of full-length Tau 4R2N trimer protease
A tau protease variant peptide or polypeptide has serine protease activity at least 95% of the full-length Tau 4R2N trimer protease.
Across the independent claim and its dependent refinements, coverage is directed to tau protease variant peptides or polypeptides with specified substitution positions in SEQ ID NO:6 that enhance serine protease activity when expressed in a bacterial host cell. Additional dependent limitations include specific permissible substitution change types and quantitative activity thresholds relative to the full-length Tau 4R2N trimer protease.
Stated Advantages
Enhanced serine protease activity compared to the original, un-substituted tau protease.
Serine protease activity levels of the tau protease variants can be quantified relative to full-length Tau 4R2N trimer protease.
Tau protease activity can be used for tau protease detection and diagnostic use in the context described, including immunoassays and activity-based assays with cleavable tau peptide substrates.
Enhanced serine protease activity compared to the original, un-substituted tau protease when expressed in a bacterial host cell.
Serine protease activity retention relative to full-length Tau 4R2N trimer protease, including activity thresholds of at least 85% and at least 95%.
Documented Applications
Diagnostic use for detecting tau protease levels or mutants associated with Alzheimer’s disease.
Immunoassay-based detection of tau protease using antibodies including N- and C-terminal antibodies.
Tau protease activity assay concept using a FRET assay with a cleavable tau peptide substrate (OP-002), including the context of inhibitor screening as described in the provided summary.
FRET-based protease activity detection using tau-cleavable fluorescent peptide substrates.
Nucleic-acid detection tools including PCR primers/probes, kits, and real-time PCR with melting curve analysis.
Diagnostic or analytical detection using antibody/immunodetection reagents for tau protease-specific epitopic core regions and immunoassays.
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