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Dimerization of Taspase1 activates an intrinsic serine protease function that leads to the catalytic Thr234 residue, which allows to catalyze the consensus sequence Q−3X−2D−1⋅G1X2D3D4, present in Trithorax family members and TFIIA. Noteworthy, Taspase1 performs only a single hydrolytic step on substrate proteins, which makes it impossible to screen for inhibitors in a classical screening approach. Here, we report the development of an HTRF reporter assay that allowed the identification of an inhibitor, Closantel sodium, that inhibits Taspase1 in a noncovalent fashion (IC50 = 1.6 μM). The novel inhibitor interferes with the dimerization step and/or the intrinsic serine protease function of the proenzyme. Of interest, Taspase1 is required to activate the oncogenic functions of the leukemogenic AF4-MLL fusion protein and was shown in several studies to be overexpressed in many solid tumors. Therefore, the inhibitor may be useful for further validation of Taspase1 as a target for cancer therapy.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
Pathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.
Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
(2021)
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.