Monitoring Enzyme Activity and Disease Biomarkers

There are four research areas in the Department of Pharmaceutical Chemistry. Monitoring enzyme activity and disease biomarkers is a research challenge within protein and cellular engineering.

The challenge

The activity of enzymes, which accelerate (catalyze) chemical reactions and alter substrates, including post-translational modifications of other proteins, is central to biological pathways (signaling, metabolism, gene regulation) and can go awry, causing disease.

Monitoring particular enzymes’ activity throughout a cell (globally)—determining its extent, rate, location, timing, and substrates amid the complex, kaleidoscopic interactivity of the proteome—is vital to determining how biological pathways normally function, detecting disease indicators (biomarkers), and thus the rational development of better targeted, more effective therapeutic interventions.

Examples of our research and methods include

Redesigning enzymes to label cleaved proteins

human caspase-7 enzyme

Depiction of human caspase-7 enzyme combines surface contours with stick features representing active site side chains: nitrogens are dark blue; sulfurs are yellow)

Molecular image made with UCSF Chimera developed by UCSF Resource for Biocomputing, Visualization, and Informatics.

Department scientists engineer proteins that monitor the activity of many enzymes, but especially proteases, which cleave the peptide bonds that link amino acids in other proteins (proteolysis), irreversibly changing them.

With about 550 types, proteases are the largest class of enzymes performing post-translational modifications in the human proteome. Their activity is vital to health (e.g., food digestion, immune response, blood coagulation) and their dysregulation underlies numerous diseases. A subset of one protease family, caspases, cleaves myriad cell proteins to bring about apoptosis, the normal programmed self-destruction of aberrant cells that fails to occur in cancers.

One approach used by researchers here is to generate antibodies via phage display that selectively bind to specific types of active proteases. The antibodies, in turn, can be linked to fluorescent compounds (immunofluorescence) so that the levels of active target proteases can be quantified by microarray (microchip-based assays).

Department scientists have also rationally redesigned a bacterial enzyme (from subtilisin to subtiligase) so that it selectively and covalently attaches biotin affinity labels to the exposed alpha amines (N-termini) of protein fragments generated by proteolytic cleavage. Thus, the fragments and their parent substrates can be globally affinity-purified, identified, and quantified via mass spectrometry.

Subtiligase molecule

Subtiligase molecule as determined by x-ray crystallography. Purple ball-and-stick figures represent bound substrate that the engineered enzyme acts on. The mutated active site residues—a serine replaced by cysteine; and proline by alanine—are labeled and highlighted in cyan (bright blue).

Such protein-based methods can be used to determine the roles of specific proteases in vital biological pathways as well as to diagnose disease by detecting and quantifying levels of biomarker enzyme activity. The latter can, in turn, help to rapidly determine, at the molecular level, if a given treatment is effective.

Our applications of such protein engineering include

Tracking blood-borne biomarkers

Using the rationally re-designed enzyme subtiligase to label N-terminal peptides from substrates cleaved by apoptotic caspases in three different blood cancer cell types treated with three different cytotoxic drugs. This approach potentially provides cell-type specific biomarkers of drug action and efficacy. In addition, common caspase substrates among different cells and in response to different drugs suggest key apoptotic nodes for further drug targeting.

engineered enzyme labels and isolates N-terminal peptides

Using an engineered enzyme (subtiligase) to label and isolate N-terminal peptides in complex mixtures, including human blood, thus revealing substrates and quantifying activity levels of proteolytic enzymes, which may be biomarkers for disease.

From the top:

  1. A synthesized peptide combines biotin (B) with a linker (amino acid sequence ENLYFQ) and a serine-tyrosine dipeptide (SY).
  2. Subtiligase attaches that peptide to a protein N-terminus (H2N) N-terminus amine group.
  3. Biotin binds to streptavidin-coated beads (avidin).
  4. The digestive enzyme trypsin is used to cleave the protein from its carboxyl side.
  5. A non-human protease from the tobacco etch virus (TEV) selectively targets and cleaves away the linker sequence.
  6. This leaves the SY-dipeptide tag on the N-terminus, which is isolated by chromatography based on electrical charge (SCX Fractionation) for analysis by tandem mass spectrometry (MS/MS).

Ongoing research seeks to develop a quantitative platform to monitor caspase proteolytic output in patients being treated for blood cancers. The hypothesis is that certain cell-type malignancy-specific protein fragments released when cancer cells are driven to apoptosis by chemotherapy will allow for rapid, real-time monitoring of serum biomarkers of a given treatment’s efficacy. Creating antibodies for the proteolytic fragments via phage display would enable high-throughput quantification of such biomarkers.