Proteolytic Systems and Types of Extracellular Substrates

In macrophages, specific receptors usually recognize a specific type of substrate upon contact by which a signaling cascade is triggered to initiate a specific response. Often, different receptor systems collaborate or compete in substrate recognition [52]. While there are cases for receptor-independent uptake, the uptake of larger particles is usually initiated by receptors. For the uptake of extracellular oxidized protein compounds, the group of macrophagial

"scavenger receptors" are of particular interest. In particular, the scavenger receptor CD36 has been identified as having a major role in the uptake of oxidized protein substrates [68].

Once internalized, compounds are enveloped in the endosome, the vesicle that becomes a lysosome as its internal pH is lowered [56]. Next to the lysosome, the other important proteolytic system is the proteasomal system. The catalytic core of the proteasome enzyme (20S proteasome) is comprised of two adjacent heptameric rings of catalytic subunits capped on either end by two further heptameric rings of regulatory subunits [11]. The 20S proteasome preferentially degrades oxidatively modified and denatured forms of protein [61]. While it had been known that intracellular proteins are degraded by the 20S proteasome [reviewed by 30], we could confirm this for internalized oxidized proteins from extracellular space [67], internalized protein from apoptotic cells [68] and for internalized glycated protein [Stolzing & Grune, in preparation].

Accumulation of extracellular modified proteins is involved in several pathologies such as arteriosclerosis, cataract formation and aging [14,32]. The accumulation of oxidized proteins can result from several kinds of malfunctions of cellular metabolism including an age-related over-expression of protein, decrease of proteolytic activities or increase of oxidative stress [31].

Extracellular proteins might be either oxidized pure proteins, glycoxidized proteins or also proteins in a more complex environment, e.g. in contact with lipids and membranes.

Glucose in a process called glycation might modify proteins. In this is a non-enzymatic reaction between the carbonyl groups of a protein and a reducing sugar various sugars might react, glucose is just the highest concentrated in the extracellular space. Stable end-products of diverse oxidation and dehydration steps, resulting from the interaction between protein and sugar lead to products called advanced glycation endproducts (AGE) [22,23]. On the other hand oxidation leads also to a modification of several amino acid side chains. Modifications are very common on the amino acid side chains leucine, valine, isoleucine or proline. These are modified to become carbonyls [13]. For the study of microglial function any protein that naturally occurs in the brain is of interest as a substrate. The Myelin Basic Protein (MBP, 18.5 kDa) is a major part of the myelin sheet of oligodendrocytes [38] and therefore, a typical brain protein.

Apoptosis of neuronal and other cells is accompanied by oxidative modification of in-tracellular proteins. Apoptotic vesicles are derived from late apoptotic cells exposed to apoptotic stimuli for a longer time, resulting in cells with decreased volume and DNA fragmentation [39]. They have an average size of 3-7^m and are therefore much smaller than cells. Additionally these vesicles have a higher proportion of sugar-structures on the membrane, which are exposed late in apoptosis [19].

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