The importance of the intramembrane protease SPPL2a for the development of murine B lymphocytes

In dieser Arbeit wurde die physiologische Bedeutung der Intramembranproteasen Signal peptide peptidase like 2a und 2b (SPPL2a und SPPL2b) mit Hilfe SPPL2a- und SPPL2b-defizienter Mäuse untersucht. Die invariante Kette (CD74), ein Chaperon von MHCII Molekülen, konnte als erstes in vivo validiertes Substrat von SPPL2a identifiziert werden. Aufgrund fehlender Intramembranproteolyse von CD74 in SPPL2a-defizienten B-Lymphozyten kommt es zur Akkumulation eines N-terminalen Fragmentes von CD74. Die Auswirkungen dieser Akkumulation auf die Entwicklung und Funktionalität der B-Zellen und die humorale Immunantwort werden beschrieben

The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

Peer reviewe

Signal-peptide-peptidase-like 2a is required for CD74 intramembrane proteolysis in human B cells

The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalysed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a(−/−) mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a(−/−) mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept

Aldosterone exerts anti-inflammatory effects on LPS stimulated microglia

Over the last years, studies on microglia cell function in chronic neuro-inflammation and neuronal necrosis pointed towards an eminent role of these cells in Multiple Sclerosis, Parkinson's and Alzheimer's Disease. It was found, that microglia cell activity can be stimulated towards a pro- or an anti-inflammatory profile, depending on the stimulating signals. Therefore, investigation of receptors expressed by microglia cells and ligands influencing their activation state is of eminent interest.A receptor found to be expressed by microglia cells is the mineralocorticoid receptor. One of its ligands is Aldosterone, a naturally produced steroid hormone of the adrenal cortex, which mainly induces homeostatic and renal effects. We evaluated if the addition of Aldosterone to LPS stimulated microglia cells changes their inflammatory profile.Therefore, we assessed the levels of nitric oxide (NO), iNOS, IL-6, IL-1β, TNF-α and COX-2 in untreated, LPS-treated and LPS/Aldosterone-treated microglia cells. Furthermore we analyzed p38-MAP-Kinase and NFκB signaling within these cells.Our results indicate that the co-stimulation with Aldosterone leads to a decrease of the LPS-induced pro-inflammatory effect and thus renders Aldosterone an anti-inflammatory agent in our model system

The intramembrane proteases signal peptide peptidase-like 2a and 2b have distinct functions in vivo

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system

Meprin beta cleaves TREM2 and controls its phagocytic activity on macrophages

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin beta is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC-MS/MS analysis of recombinant TREM2 incubated with meprin beta revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin beta cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b(-/-) mice compared to WT controls. Isolated BMDMs from Mep1b(-/-) mice showed significantly increased full-length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin beta deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin beta is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2