No abstract

The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Singlet oxygen ( 1 O 2 ) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates 1 O 2 by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. 1 O 2 oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by 1 O 2 at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by 1 O 2 . Because 1 O 2 is difficult to detect in cells, identifying oxidized cellular products uniquely derived from 1 O 2 could serve as a marker of its presence. In the present study, 1 O 2 reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to 1 O 2 generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of 1 O 2 in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest the protein environment can affect the oxidation pathway.

Abstract10-N-nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipindeficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.