Mechanisms and Function of Intramembrane Proteolysis by the gramme-Secretase homologous Signal Peptide Peptidase-like Proteases (SPPL)
Final Report Abstract
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.
Publications
- A γ-secretase-like intramembrane cleavage of TNFα by the GxGD aspartyl protease SPPL2b. Nat Cell Biol. 2006;8(8):894-6
R. Fluhrer, G. Grammer, L. Israel, MM. Condron, C.Haffner, E. Friedmann, C. Böhland, A. Imhof, B. Martoglio, DB. Teplow and C. Haass
- Signal Peptide Peptidases and γ-Secretase: Cousins of the Same Protease Family? Neurodegener Dis. 2007;4(2-3):112-116
R. Fluhrer and C. Haass
- Intramembrane Proteolysis by γ-secretase. J Biol Chem. 2008;283(44):29627-31
H. Steiner, R. Fluhrer and C. Haass
- Intramembrane Proteolysis of GxGD-type Aspartyl Proteases is slowed by a Familial Alzheimer Disease-like Mutation. J Biol Chem. 2008;283(44):30121-8
R. Fluhrer, A. Fukumori, L. Martin, G. Grammer, M. Haug-Kröper, B. Klier, E. Winkler, E. Kremmer, MM, Condron, DB. Teplow, H. Steiner and C. Haass
- Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/b. J Biol Chem. 2008;283(3):1644-52
L. Martin, R. Fluhrer, K.Reiss, E. Kremmer, P.Saftig and C. Haass
- Intramembrane Proteolysis by Signal Peptide Peptidases – A Comparative Discussion of GxGD-Type Aspartyl Proteases. J Biol Chem. 2009 May 22;284(21):13975-9
R. Fluhrer, H. Steiner, and C. Haass
- Intramembrane Proteolysis by γ-Secretase and Signal Peptide Peptidases. In: Research and Perspectives in Alzheimer's Disease - Intracellular Traffic and Neurodegenerative Disorders Springer Verlag, Berlin Heidelberg 2009
R. Fluhrer and C. Haass
- Substrate Requirements for SPPL2b Dependent Regulated Intramembrane Proteolysis. J Biol Chem. 2009;284(9):5662-70
L. Martin, R. Fluhrer and C. Haass
- The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain. J Exp Med. 2013 Jan 14;210(1):41-58
J. Schneppenheim, R. Dressel, S. Hüttl, R. Lüllmann-Rauch, M. Engelke, K. Dittmann, J. Wienands, EL. Eskelinen, I. Hermans-Borgmeyer, R. Fluhrer, P. Saftig, B. Schröder
- Three-amino acid spacing of presenilin endoproteolysis suggests a general stepwise cleavage of gamma-secretase-mediated intramembrane proteolysis. J Neurosci. 2010 Jun 9;30(23):7853-62
A. Fukumori, R. Fluhrer, H. Steiner and C. Haass
- Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3). J Biol Chem. 2012 Dec 21;287(52):43401-9
M. Voss, A. Fukumori, P-H. Kuhn, U. Künzel, B. Klier, G. Grammer, M. Haug-Kröper, E. Kremmer, SF. Lichtenthaler, H. Steiner, B. Schröder, C. Haass and R. Fluhrer
(See online at https://doi.org/10.1074/jbc.M112.371369) - Palmitoylation of TNF alpha is involved in the regulation of TNF receptor 1 signalling. Biochim Biophys Acta. 2012 Nov 16. S0167-4889(12)00330-8
M. Poggi, I. Kara, JM. Brunel, JF. Landrier, R. Govers, B. Bonardo, R. Fluhrer, C. Haass, MC. Alessi, F. Peiretti
(See online at https://doi.org/10.1016/j.bbamcr.2012.11.009) - The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b). J Biol Chem. 2012 Feb 10;287(7):5156-63
R. Fluhrer, L. Martin, B. Klier, M. Haug-Kröper, G. Grammer, B. Nuscher and C. Haass
(See online at https://doi.org/10.1074/jbc.M111.328104) - The intramembrane protease SPPL2A is critical for tooth enamel formation. J Bone Miner Res. 2013 Feb 20
AL. Bronckers, N. Güneli, R. Lüllmann-Rauch, J. Schneppenheim, AP. Moraru, N. Himmerkus, TJ. Bervoets, R. Fluhrer, V. Everts, P. Saftig, B. Schröder
(See online at https://doi.org/10.1002/jbmr.1895) - The transferrin receptor-1 membrane stub undergoes intramembrane proteolysis by signal peptide peptidase-like 2b. FEBS J. 2013 Apr;280(7):1653-63
C.Zahn, M. Kaup, R. Fluhrer, H. Fuchs
(See online at https://doi.org/10.1111/febs.12176)
