Daniela Stauffer

SPPL2a and SPPL2b promote intramembrane proteolysis of TNFα in activated dendritic cells to trigger IL-12 production

Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFα). The two proteases promoted the release of the TNFα intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.

PROTEIN ISOASPARTYL METHYLTRANSFERASE PROTECTS FROM BAX-INDUCED APOPTOSIS

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinsons drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

Discovery of novel indolinone-based, potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinsons disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.

Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα) are both transcriptional regulators of the Runx2-I isoform.

Runx2 is a master regulator of bone development and has also been described as an oncogene. Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα), both implicated in bone metabolism and breast cancer, have been shown to share common transcriptional targets. Here, we show that ERα is a positive regulator of Runx2-I transcription. Moreover, ERRα can act as a transcriptional activator of Runx2-I in presence of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α). In contrast, ERRα behaves as a negative regulator of Runx2-I transcription in presence of PGC-1β. ERα and ERRα cross-talk via a common estrogen receptor response element on the Runx2-I promoter. In addition, estrogen regulates PGC-1β that in turn is able to modulate both ERα and ERRα transcriptional activity.

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of autosomal‐dominant forms of Parkinsons disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein‐protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule‐associating protein (MAP)/microtubule affinity‐regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT‐binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic‐labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.—Krumova, P., Reyniers, L., Meyer, M., Lobbestael, E., Stauffer, D., Gerrits, B., Muller, L., Hoving, S., Kaupmann, K., Voshol, J., Fabbro, D., Bauer, A., Rovelli, G., Taymans, J.‐M., Bouwmeester, T., Baekelandt, V. Chemical genetic approach identifies microtubule affinity‐regulating kinase 1 as a leucine‐rich repeat kinase 2 substrate. FASEB J. 29, 2980‐2992 (2015). www.fasebj.org

Blockade of Metallothioneins 1 and 2 Increases Skeletal Muscle Mass and Strength

ABSTRACT Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo. We found that concomitant abrogation of metallothioneins 1 and 2 results in activation of the Akt pathway and increases in myotube size, in type IIb fiber hypertrophy, and ultimately in muscle strength. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function was also observed in the setting of glucocorticoid addition, which is a strong atrophy-inducing stimulus. Given the blockade of atrophy and the preservation of strength in atrophy-inducing settings, these results suggest that blockade of metallothioneins 1 and 2 constitutes a promising approach for the treatment of conditions which result in muscle atrophy.