Resistencia y durabilidad

As BACE is the rate limiting step of Amyloid-β production, interactions of BACE as with other possibly regulating proteins are of great interest.

Among the AD related proteins, it was proposed that Nicastrin co-immunoprecipitates with BACE and also increases β-secretase activity in COS-7 cells, as measured by amount of soluble APP produced 204. Also, a direct binding between BACE and PS1 was suggested as shown by co-localization studies in human cultured cells as well as coimmunoprecipitation that preferentially detected immature BACE, leading to the author’s hypothesis, that Presenilin might potentially act as a regulator of BACE maturation 205. In addition, a study presented data that the ectodomain of BACE, when exogenously added to cells expressing APP, can interact with the ectodomain of APP on the cell surface leading to its internalization. This interaction does not seem to be dependent on substrate binding to the active site of BACE, since neither antibodies direct

against the active site nor competitive inhibitors could decrease internalization. Also, co- immunoprecipitation of full length BACE and APP from lysates was shown 206.

The C-terminus of BACE possibly interacts with proteins of the cellular trafficking machinery like the GGA proteins 147, 148, which may regulate the phosphorylation dependent endocytosis of BACE 144 as presented above in the section on BACE trafficking. In addition, binding of the cytoplasmic region of BACE, likely the Di-leucine motif, to the C-terminus of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, was shown by Co-immunoprecipitation and Co-localization with buoyant lipid microdomains in SH-SY5Y cells 207. The authors propose that by this mechanism BACE could be recruited into detergent-insoluble lipid rafts.

A yeast two-hybrid system furthermore identified the brain-specific type II membrane protein BRI3 as an interaction partner with the C-terminal cytosolic tail of BACE, which interestingly is also converted by Furin as is BACE 208_.

Reference Substrate Sequence

Enzyme type Method KM

(µM) Kcat (min- 1) Kcat / KM (µM-1 * min- 1) “Swedish-like” substrates 209 _{Substrate„FS-2“:} (MCA)SEVNL*DAEFK( DNP) BACE-NT Fluorogenic 4,5 0,25 0,056 186 _{Substrate „SW“:} EVNL*DAEFWHDR BACE-NT MALDI-TOF 35,8 54,7 1,528

137 _{SEVNL*DAEFR BACE-NT Fluorogenic 9 1,2 0,133}

BACE-FL not speci- fied not speci- fied

210 _{AcEVNL*DAEFK(DNP) BACE-NT from}

S2 insect cells FRET- methoxycum arin group n.d. n.d. 0,004 “” BACE-NT from CHO cells n.d. n.d. 0,005 “” BACE-FL from HEK cells n.d. n.d. 0,003 185 _{SEVNL*DAEFR HPLC 1000 2,45 147} 211 _{AcEVNL*DAEFK(DNP) Reverse} Phase Column n.d. n.d. 0,008 212 _{TEEISEVNLDAEFRHD} SGK truncated PreProBACE (amino acids 1– 460) insect cells reverse- phase HPLC 186600 “” PreProBACE treated with Furin to generate mature BACE 438000

Substrate with „optimized“ sequences

213 _{Substrate “OK“:} EIDL*MVLDWHDR HPLC 5,2 19,2 3,692 186 _{Substrate “OK“:} EIDL*MVLDWHDR MALDI-TOF 1,4 28,6 20,429

Figure 13: Overview of kinetic parameters of BACE from the literature

The entries of the table are categorized into “swedish-like substrates” and “optimized sequences”. The BACE cleavage site is indicated by an asterisk *.

6 Goals of the current work

BACE cleavage is the rate limiting step in generating Amyloid-β. Since the identification of BACE, important knowledge, of its tissue expression and regulation, its maturation and trafficking, its substrate profile and putative interaction partners has been generated. However, attempts to inhibit this enzyme based on information from crystal structures of the soluble BACE ectodomain encounter the difficulty that BACE seems to have an unusually wide cleft of its active center. It is thus of paramount interest to study the conformation and the enzymatic characteristics of BACE in its native configuration. The present work thus aimed at clarifying whether native BACE functions in a complex or in cooperation with other proteins and whether this might influence its physiological function. This knowledge could have important impact on our understanding of the physiologic function of BACE with respect to its interaction with substrates and the regulation of its enzymatic activity. In addition these insights might open alternative pathways for therapeutic intervention.

For this purpose the following specific aims were formulated:

1) Development of an appropriate method to study the native conformation of BACE a. In particular it was intended to adapt Blue-Native Polyacrylamide

Electrophoresis (BN-PAGE), which was successfully used for the study the

γ-secretase complex 93, 214_{, for the study of native BACE. To this end, a} method for native preparation of protein complexes for BACE derived from neuronal tissues and cultured cells had to be optimized. Moreover, the BN- PAGE separation technique should be improved for BACE and a detection of native BACE by Western Blot with an appropriate sensitivity for endogenous levels should be achieved.

b. The native size of BACE should consequently be identified in native samples from different tissues and cell lines in comparison to the size of BACE under denaturating conditions on SDS-PAGE.

2) Characterization of the quaternary structure of BACE

a. The putative oligomeric structure of native BACE should be analyzed by appropriate methods such as co-immunoprecipitation studies.

b. The posttranslational modification of native BACE within the putative complex should be identified.

3) Differences in quaternary structure with respect to subcellular localization

a. Subcellular sites of assembly of the putative BACE complex should be identified with appropriate constructs containing retention motifs which control the subcellular localization of the corresponding proteins.

4) Identification of domains within BACE involved in the putative binding interface a. A domain deletion analysis should be performed by generating appropriate

constructs with intact enzymatic function. The native size of the constructs should subsequently be analyzed by BN-PAGE.

5) Investigation of the potential physiological role of a putative BACE complex

a. A putative influence of the native quaternary conformation on stability, trafficking and subcellular localization should be investigated in comparison to the properties of BACE under denaturating conditions.

b. With respect to the fact that BACE is the rate-limiting enzyme for the generation of Aβ, it was furthermore an intended goal of this study to characterize the enzymatic properties of the putative native BACE complex in vivo and in vitro.

7 Material and Methods