Expanded Interactome of the Intrinsically Disordered Protein Dss1

Summary: Dss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome. : Schenstrøm et al. demonstrate that the disordered protein Dss1 forms a transient intramolecular interaction between the C-terminal helical region and a central hydrophobic region. Proteomics reveal several Dss1-binding proteins, including all PCI-domain protein complexes. The dynamic fold-back structure regulates Dss1 interactions with the mitotic septins and ATP-citrate lyase. Keywords: intrinsically disordered proteins, protein degradation, proteasome, PCI domai

The human Na⁺/H⁺ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

Background Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. Methods and results Here, we identify the human Na+/H+ exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner. Conclusions This work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2

Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations

Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin

Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na+/H+-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of pT779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.Lundbeck Foundation; National Institute of Neurological Disorders and Stroke [R01NS091336]; National Institute of General Medicine [R01GM098482]; Danish Council for Independent Research Natural Sciences [4181-00344]; Novo Nordisk Foundation [NNF18OC0034070]; US Department of Energy, Office of Science and Office of Basic Energy Sciences [DE-AC02-76SF00515]; DOE Office of Biological and Environmental Research; National Institutes of Health, National Institute of General Medical Sciences [P41GM103393]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Escape from the chains of sense: towards the subversive force of the texts of F. M. Dostojevsky, F. Kafka and W. Gombrowicz

My thesis "Escape from the Chains of Sense: Towards the Subversive Force of the Texts of F. M. Dostoevsky, F. Kafka and W. Gombrowicz" focuses on the phenomenon here called the f1ight of sense. This notion challenges interpretation as a desire for order and for synthesis of the work's semantic direction, and caUs for a different reading of the work. In the introduction, using Adorno's, Deleuze's and Guattari' s concepts, I try to show that the f1ight of sense is not a final state (being without sense) but the act of its f1eeing. Inf1uenced by Barthes and his concept of the pleasure ofthe text, I call the spaces ofthe subversive force, which lets the sense and its reconstruction slip away, ruptures. They lead to a "creative rnisunderstanding" which is a ground for experirnentation. This experimentation does not refuse interpretation and its hermeneutic claim on clear understanding but suggests a different approach: a reading that looks through these ruptures and lets itself get seduced by the gestures and ornaments, which defer or dissolve meaning in the text s of Do sto evsky, Kafka and Gombrowicz. Thus experimentation is an attempt to describe how these elements work together

The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins