Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation

Parvulin 17-catalyzed tubulin polymerization is regulated by calmodulin in a calcium-dependent manner

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTPdependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca2+-loaded calmodulin (Ca2+/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca2+/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca2+/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with 15N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca2+-dependent manner with the Par17 N terminus. The reverse experiment with 15N-labeled Ca2+/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca2+/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK796-815 complex. In vitro tubulin polymerization assays furthermore showed that Ca2+/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca2+/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca2+/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca2+ signaling with microtubule function.Fil: Burgardt, Noelia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Schmidt, Andreas. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Manns, Annika. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Schutkowski, Alexandra. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Jahreis, Günther. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Lin, Yi Jan. Kaohsiung Medical University; AlemaniaFil: Schulze, Bianca. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Masch, Antonia. Martin Luther University Halle Wittenberg; AlemaniaFil: Lücke, Christian. Max Planck Research Unit for Enzymology of Protein Folding; AlemaniaFil: Weiwad, Matthias. Max Planck Research Unit for Enzymology of Protein Folding; Alemania. Martin Luther University Halle Wittenberg; Alemani

Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38*

Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor α subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor α regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membrane- anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase