ERANET_NEURON_1. Call_Role of proteases and their inhibitors in the pathophysiology and diagnosis of Alzheimer Disease (ADTest)

The pathological hallmark of Alzheimer`s disease (AD) is the formation of amyloid plaques in the extracellular (EC) matrix of the brain. It has been shown that multiple proteases act cooperatively in regulating the steady-state levels of brain amyloid ß-peptide (Aß), the main constituent of the amyloid plaques. The activity of these proteases depends on proteolytic activation and interaction with endogenous inhibitors like the proteoglycan testicans 1-3, multidomain EC proteins comprising different protease inhibitor modules, each one directed against a different kind of protease. These findings suggest that senile plaque formation is subject to a fine-tuned interplay of proteases and cognate inhibitors, which in concert determine the rates of Aß degradation and aggregation and thus the progression of the AD disease. To better understand the underlying regulatory mechanisms we aim to perform a structure-function analysis of the inhibitor domains of testicans 1-3 and study complex formation with proteases implicated in amyloid removal. Notably, also other EC proteoglycans in the brain such as neurocan, versican and perlecan contain similar regulatory domains. Therefore, the uncovered regulatory mechanism employed by testican might serve as a paradigm for EC protease modulation that should be relevant in general for protein folding diseases and might guide the development of novel medical strategies.

Alzheimers disease (AD) is caused by abnormally folded A? and Tau proteins, which can aggregate in the brain as extracellular amyloid plaques or intracellular neurofibrillary tangles, respectively. Since plaque and fibril formation advance in a concentration-dependent manner, the quality control of soluble and aggregated cerebral proteins is of major importance for the development of AD. It has been shown that activated astrocytes surrounding amyloid plaques control this process by secreting dedicated proteases to degrade A?. Notably, the activity of the secreted proteases is fine-tuned by sophisticated regulatory mechanisms including proenzyme activation and allosteric modulation. In addition, specific endogenous inhibitors, which belong to the so-called testican family, modulate the maturation and activity of cerebral proteases and thus function as important regulators of A? metabolism. To better understand the fine-tuned interplay of proteases and cognate inhibitors during the progression of AD, we initiated a structure-function analysis of the testican proteins and their targeted proteases belonging to the HtrA protease family. Owing to problems in producing the testican inhibitor in recombinant form, we focused our work on characterizing the human HtrA1 protease and its bacterial counterpart DegQ.Mammalian HtrA1 is a PDZ-containing protease that is ubiquitously expressed playing an important physiological role in the extracellular matrix. Moreover, it is associated with various protein folding diseases such as AD, arthritis and age-related macular degeneration. Despite its importance, little is known about the proteolytic mechanism of HtrA1. Within this project, we crystallized human HtrA1 in its active and inactive conformations and performed biochemical analyses addressing its mechanistic features. In contrast to related proteases, the activity of the HtrA1 protease is directly controlled by induced-fit substrate binding and does not depend on allosteric ligands acting in trans. This auto-activation mechanism provides an unprecedented regulatory facet of HtrA proteases and implies that HtrA1 is under the control of further protein-protein interactions that influence its specific localization and/or its substrate binding properties. The obtained findings provide the molecular basis to address these processes and evaluate the precise function of HtrA1 under normal and pathological conditions.In an extension of the HtrA1 project, we characterized the bacterial DegQ protease-chaperone. The major aim was to address how DegQ functions in an ATP-independent manner, i.e. without using chemical energy. Our structural and biochemical data imply that DegQ and functionally related ATP-consuming quality control factors employ similar mechanistic strategies to capture and remodel client proteins. However, the energy to either refold or degrade the encapsulated clients is derived from the substrate-induced oligomer conversion. This mechanism appears to be broadly relevant for HtrA proteins and for other proteins operating in the extracellular space that is devoid of ATP.