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Robert L Heinrikson

Publications and source records attributed to Robert L Heinrikson.

4 recordsLinked to original sources
PubMed2005

An efficient method to express and refold a truncated human procaspase-9: a caspase with activity toward Glu-X bonds.

A truncated form of human procaspase-9 missing the first 111 amino acids, and a variety of mutants derived therefrom, have been expressed in Escherichia coli inclusion bodies. Upon refolding to active enzymes, Delta(1-111) procaspase-9 and mutants were recovered at purity greater than 95% and with a final yield of 20-35 mg/L cell culture. Our active procaspase-9 retains its pro-segment, while undergoing major auto processing at Asp315 and a minor (20%) cleavage at Glu306. This unusual cleavage at a Glu-X bond also took place in the D315E mutant, and we describe herein the inhibitor Z-VAE-fmk that shows enhanced inactivation of procaspase-9 over caspases-3. The bond at Asp330, not processed by procaspase-9, is cleaved by caspase-3 and the resulting procaspase-9 variant, missing the 316-330 bridge, is six times as active as the non-mutated Delta(1-111) proenzyme. A deletion mutant lacking residues 316-330 underwent auto activation by cleavage at Asp315-Ala331 bond. Moreover, substitution of Glu306 by an Asp residue in this mutant led to rapid removal of the peptide spanning Ser307 to Asp330, and resulted in an enzyme that was 7.6 times as active as the non-mutated Delta(1-111) procaspase-9. Finally, replacing both Asp315 and Glu306 with Ala generated a procaspase-9 mutant incapable of auto processing. This single chain procaspase-9 was fully as active as the non-mutated Delta(1-111) enzyme processed at Asp315 or Glu306. Our demonstration that unprocessed procaspase-9 mutants are active as proteases with caspase-type specificity suggests that the role of procaspase-9 in cascade activation of executioner caspases might, in some circumstances, be carried out alone and without association of the apoptosome.

Amino Acid Metabolism, Inborn Errors↗
PubMed2003

Heterodimer formation is essential for heparanase enzymatic activity.

Heparanase is an endo-beta-D-glucuronidase involved in cleavage of heparan sulfate residues and hence participates in extracellular matrix degradation and remodeling. The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a approximately 65 kDa band in SDS-PAGE analysis. The protein undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, Glu(109)-Ser(110) and Gln(157)-Lys(158), yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus, and a 6 kDa linker polypeptide that resides in-between. The active form of heparanase has long been thought to be a 50 kDa polypeptide isolated from cells and tissues. However, attempts to obtain heparanase activity after expression of the 50 kDa polypeptide failed, suggesting that the N-terminal region is important for heparanase enzymatic activity. It has been hypothesized that heterodimer formation between the 8 and 50 kDa heparanase subunits is important for heparanase enzymatic activity. By individually or co-expressing the 8 and 50 kDa heparanase subunits in mammalian cells, we demonstrate specific association between the heparanase subunits by means of co-immunoprecipitation and pull-down experiments. Moreover, a region in the 50 kDa heparanase subunit that mediates interaction with the 8 kDa subunit was identified. Altogether, our results clearly indicate that heterodimer formation is necessary and sufficient for heparanase enzymatic activity in mammalian cells.

Amino Acids↗
PubMed2003

Employing a superior BACE1 cleavage sequence to probe cellular APP processing.

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.

Alzheimer Disease↗