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Mechanisms and Function of Intramembrane Proteolysis by the gramme-Secretase homologous Signal Peptide Peptidase-like Proteases (SPPL)

Fachliche Zuordnung Molekulare Biologie und Physiologie von Nerven- und Gliazellen
Förderung Förderung von 2006 bis 2014
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 25923830
 
Erstellungsjahr 2013

Zusammenfassung der Projektergebnisse

Intramembrane Proteolysis has become subject of intensive research, since intramembrane proteases as well as their substrates have been shown to be involved in pivotal physiological and pathophysiological processes, like regulation of cholesterol metabolism, development of Alzheimer disease rheumatoid arthritis and Morbus Chron, hepatitis C infections and many more. Signal peptide peptidase (SPP), its homologues the SPP-like (SPPL) proteases and presenilin, which form the active subunit of the γ-secretase complex, are intramembrane cleaving aspartyl proteases harboring a conserved GxGD motif in one of their transmembrane domains. In humans four SPPL proteases (SPPL2a, SPPL2b, SPPL2c and SPPL3) have been identified using computational analysis. However, their physiological function and the mechanism by which these enzymes cleave their substrates were completely unknown. Moreover, it was well established that γ-secretase inhibitors, developed to slow progression of Alzheimer's disease, also target SPP and thus likely also its homologues the SPPL proteases. This project therefore aimed to shed light on the biological function of SPPL proteases and the mechanism by which these proteases process their substrates. By candidate approach we identified tumor necrosis factor alpha (TNFα) and Bri2 (Itm2b), which is associated with familial British and Danish Dementia, as the first substrates for intramembrane cleavage by SPPL2a and SPPL2b. In addition we demonstrate that the foamy virus envelope protein (FVenv) is subject to intramembrane proteolysis by SPPL3 in a cellular model system. Thus FV(env) is the first intact transmembrane protein shown to undergo cleavage by SPPL3. Detailed analysis of the SPPL2b cleavage sites within TNFα suggests that SPPL proteases utilize multiple intramembrane cleavages to liberate the hydrophobic transmembrane domains of their substrates from the cellular membrane. A similar cleavage mechanism has been described for γ-secretase. Determination of the C-terminal SPPL cleavage site within transferrin receptor 1, another SPPL2a/b substrate, further supports this cleavage model. Similar to γ-secretase, SPPL2b preferentially cleaves substrates with an ectodomain shorter than 60 amino acids. This finding in particular was surprising, since the selectivity of γ-secretase towards substrates with a short ectodomain is attributed to its co-factor Nicastrin but SPPL proteases have been shown to be catalytically active without any co-factors. Substrate recognition by SPPL2b, however, is not solely triggered by a short ectodomain of the substrate. In addition certain primary sequence determinants within the juxtamembrane and transmembrane domains of the substrate are required for efficient processing. In particular helix destabilizing amino acids within the transmembrane domain of the substrate facilitate cleavage by SPPL2b. Contrary to our expectations, only one out of four conserved amino acids that potentially destabilize α-helical transmembrane domains indeed reduced the γ-helical content of the Bri2 transmembrane domain and thus blocked its cleavage by SPPL2b. Surprisingly, SPPL3 also accepts substrates with significantly longer ectodomains as demonstrated by the use of a mutant FVenv. Thus SPPL3 is the first intramembrane aspartyl protease that is capable of processing its substrates independent of a preceding shedding event, thus acting itself like a sheddase, similar to members of the rhomboid family of intramembrane cleaving serine proteases. The physiological function of SPPL proteases is still hardly known. However, SPPL2a -/- mice suffer from a significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses due to impaired processing of the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII), highlighting SPPL2a as a potential pharmacological target for depleting and/or modulating B cells. The results that emerge provide a first insight into the cleavage mechanism, inhibitor sensitivity and physiological function of SPPL proteases and uncover certain similarities and differences between the members of the GxGD protease family. These findings represent the basis to understand the biochemical diversity and the biological significance of SPP/SPPL intramembrane proteases as well for the development of specific inhibitors. A central future issue will be to extend and define the substrate spectrum of each individual protease and to verify their physiological relevance in order to fully understand the biological function of SPP/SPPL proteases.

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