Applications of Unnatural Amino Acids in Protease Probes.

Since proteases are involved in a wide range of physiological and disease states, the development of novel tools for imaging proteolytic enzyme activity is attracting increasing interest from scientists. Peptide substrates containing proteinogenic amino acids are often the first line of defining enzyme specificity. This Minireview outlines examples of major recent advances in probing proteases using unnatural amino acid residues, which greatly expands the possibilities for designing substrate probes and inhibitory activity-based probes. This approach already yielded innovative probes that selectively target only one active protease within the group of enzymes exhibiting similar specificity both in cellular assays and in bioimaging research.

Fluorescent activity-based probe for the selective detection of Factor VII activating protease (FSAP) in human plasma.

The zymogen form of circulating Factor VII activating protease (FSAP) is activated by histones that are released as a consequence of tissue damage or excessive inflammation. This is likely to have consequences in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To investigate the existence, as well as the concentration of active FSAP (FSAPa) in complex biological systems an active site probe is needed. We used Hybrid Combinatorial Substrate Library (HyCoSuL) to screen for natural and unnatural amino acids that specifically bind to P4-P2 pockets of FSAPa. This information was used to designing a fluorogenic substrate (Ac-Pro-DTyr-Lys-Arg-ACC) as well as an irreversible, fluorogenic activity-based probe Cy5-6-Ahx-Pro-DTyr-Lys-ArgP(OPh)2. In normal human plasma the probe showed very low non-specific reactivity with some plasma proteins but upon activation of pro-FSAP with histones, strong labelling of FSAPa was observed. This labelling could be inhibited by aprotinin and was not found in the plasma of a subject that was homozygous for a polymorphism, which leads to loss of activity, or in plasma that was depleted of FSAP by antibodies. This 2nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1st generation substrate and a much higher selectivity for FSAPa over other plasma proteases. This substrate and probe can be useful to detect and localize FSAPa in normal and pathological tissue and plasma to gain more insight into its functions.

[Biochemical markers for Alzheimer disease]. / Biokemiske markører for Alzheimers sygdom.

It is estimated that approximately 40,000 people in Denmark suffer from Alzheimer's disease (AD), a neurodegenerative dementia. Symptomatic treatment now exists which can temporarily inhibit the loss of brain function. Unfortunately, AD is difficult to diagnose, especially early in the disease course, and a definite diagnosis is possible only post-mortem. To develop improved diagnostic methods, several biomarkers have been examined for their ability to differentiate AD from normal aging and other dementias. Their measurement in blood samples has not yet been developed, but analyses may now be routinely performed using cerebrospinal fluid (CSF). The CSF biomarkers include amyloid-beta 1-42 (Abeta1-42), which is decreased in AD patients, as well as total tau (t-tau) and hyperphosphorylated tau (p-tau), which are elevated. While a decreased Abeta1-42 level in CSF has no independent value in the diagnostic differentiation, t-tau and p-tau add more specificity to the differentiation of AD from other dementias while retaining a reasonable degree of sensitivity.

Years of severe and isolated amnesia can precede the development of dementia in early-onset Alzheimer's disease.

A young patient with a severe and isolated memory disorder, meeting the criteria for MCI, was followed for a period of 8 years. His overall functional level remained stable with a CDR-score at 0.5 until the last year when it dropped to 1.0. Neuropsychological testing showed severe memory deficits but otherwise normal cognitive functions. Only minimal progression was measured; however, the last testing showed impaired abstraction and executive functioning as well as discrete problems generating names of objects and people. Neuroimaging, with MRI and SPECT, was consistent with a progressive degenerative disorder, and cerebrospinal fluid levels of beta-amyloid 1-42, tau protein, and phosphorylated tau protein were characteristic of Alzheimer's disease (AD). We argue that this is a case of prodromal AD, which illustrates an extreme version of the normal course with respect to slow progression of the disease and severity of amnesia early in the course.