Identification of HCN1 as a 14-3-3 client

Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. Isothermal titration calorimetry demonstrated that recombinant 14-3-3 binds all three phospho-peptides with low micromolar affinity. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two binding sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding sites is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

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Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

F1000Research ◽

10.12688/f1000research.11808.1 ◽

2017 ◽

Vol 6 ◽

pp. 1166 ◽

Cited By ~ 6

Author(s):

Olivia R. Buonarati ◽

Peter B. Henderson ◽

Geoffrey G. Murphy ◽

Mary C. Horne ◽

Johannes W. Hell

Keyword(s):

Gene Expression ◽

Brain Tissue ◽

Neuronal Excitability ◽

Central Element ◽

Proteolytic Processing ◽

Full Length ◽

Hek293 Cells ◽

Specific Antibodies ◽

C Terminus ◽

Molecular Masses

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (MR)of the α11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α11.2 expressed in HEK293 cells was conducted using six different region–specific antibodies against α11.2. Results: The full length form of α11.2 migrated, as expected, with an apparent MR of ~250 kDa. A shorter form of comparable prevalence with an apparent MR of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

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Coordination of RNA and protein condensation by the P granule protein MEG-3

10.1101/2020.10.15.340570 ◽

2020 ◽

Author(s):

Helen Schmidt ◽

Andrea Putnam ◽

Dominique Rasoloson ◽

Geraldine Seydoux

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Protein ◽

Protein Protein Interactions ◽

C Elegans ◽

Intrinsically Disordered ◽

Disordered Protein ◽

C Terminus ◽

Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

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Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

F1000Research ◽

10.12688/f1000research.11808.2 ◽

2018 ◽

Vol 6 ◽

pp. 1166 ◽

Cited By ~ 1

Author(s):

Olivia R. Buonarati ◽

Peter B. Henderson ◽

Geoffrey G. Murphy ◽

Mary C. Horne ◽

Johannes W. Hell

Keyword(s):

Gene Expression ◽

Brain Tissue ◽

Neuronal Excitability ◽

Central Element ◽

Proteolytic Processing ◽

Full Length ◽

Hek293 Cells ◽

Specific Antibodies ◽

C Terminus ◽

Molecular Masses

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Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle

Journal of Virology ◽

10.1128/jvi.74.11.5388-5394.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5388-5394 ◽

Cited By ~ 36

Author(s):

Judith A. O'Brien ◽

John A. Taylor ◽

A. R. Bellamy

Keyword(s):

Amino Acids ◽

Binding Sites ◽

Limited Proteolysis ◽

Coiled Coil ◽

Surface Binding ◽

Virus Maturation ◽

C Terminus ◽

Tetramerization Domain ◽

Necessary And Sufficient ◽

Sequence Requirements

ABSTRACT The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione S-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.

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Dri1 mediates heterochromatin assembly via RNAi and histone deacetylation

Genetics ◽

10.1093/genetics/iyab032 ◽

2021 ◽

Author(s):

Hyoju Ban ◽

Wenqi Sun ◽

Yu-hang Chen ◽

Yong Chen ◽

Fei Li

Keyword(s):

Histone Deacetylases ◽

Genome Stability ◽

Rna Binding ◽

Histone Deacetylation ◽

Rnai Pathway ◽

Chromatin Domain ◽

Heterochromatin Protein 1 ◽

Intrinsically Disordered ◽

C Terminus ◽

Heterochromatin Assembly

Abstract Heterochromatin, a transcriptionally silenced chromatin domain, is important for genome stability and gene expression. Histone 3 lysine 9 methylation (H3K9me) and histone hypoacetylation are conserved epigenetic hallmarks of heterochromatin. In fission yeast, RNA interference (RNAi) plays a key role in H3K9 methylation and heterochromatin silencing. However, how RNAi machinery and histone deacetylases (HDACs) are coordinated to ensure proper heterochromatin assembly is still unclear. Previously, we showed that Dpb4, a conserved DNA polymerase epsilon subunit, plays a key role in the recruitment of HDACs to heterochromatin during S phase. Here, we identified a novel RNA-binding protein Dri1 that interacts with Dpb4. GFP-tagged Dri1 forms distinct foci mostly in the nucleus, showing a high degree of colocalization with Swi6/Heterochromatin Protein 1. Deletion of dri1+ leads to defects in silencing, H3K9me, and heterochromatic siRNA generation. We also showed that Dri1 physically associates with heterochromatic transcripts, and is required for the recruitment of the RNA-induced transcriptional silencing (RITS) complex via interacting with the complex. Furthermore, loss of Dri1 decreases the association of the Sir2 HDAC with heterochromatin. We further demonstrated that the C-terminus of Dri1 that includes an intrinsically disordered (IDR) region and three zinc fingers is crucial for its role in silencing. Together, our evidences suggest that Dri1 facilitates heterochromatin assembly via the RNAi pathway and HDAC.

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Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/150.3.977 ◽

1998 ◽

Vol 150 (3) ◽

pp. 977-986 ◽

Cited By ~ 4

Author(s):

Yangsuk Park ◽

John Hanish ◽

Arthur J Lustig

Keyword(s):

Amino Acids ◽

Active Site ◽

Fusion Proteins ◽

Initiation Site ◽

Negative Control ◽

Model System ◽

Mutant Protein ◽

Regulatory Domain ◽

C Terminus ◽

Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

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Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

Cellular and Molecular Life Sciences ◽

10.1007/s00018-020-03639-z ◽

2020 ◽

Author(s):

Lasse Staby ◽

Katherine R. Kemplen ◽

Amelie Stein ◽

Michael Ploug ◽

Jane Clarke ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Three Dimensional ◽

Life Time ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Order And Disorder ◽

C Terminus ◽

Close Relationship ◽

Stability Dynamics ◽

And Function

Abstract Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

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Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

AJP Renal Physiology ◽

10.1152/ajprenal.00133.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. F327-F333 ◽

Cited By ~ 147

Author(s):

Rémon A. M. H. Van Aubel ◽

Pascal H. E. Smeets ◽

Jeroen J. M. W. van den Heuvel ◽

Frans G. M. Russel

Keyword(s):

Binding Sites ◽

Efflux Pump ◽

Substrate Binding ◽

Organic Anion ◽

Apical Cell ◽

Cell Uptake ◽

Hek293 Cells ◽

Hill Coefficient ◽

Anion Transporter ◽

Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

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Oligomerization of the Human Adenosine A2AReceptor is Driven by the Intrinsically Disordered C-Terminus

Biophysical Journal ◽

10.1016/j.bpj.2020.11.760 ◽

2021 ◽

Vol 120 (3) ◽

pp. 91a

Author(s):

Khanh D. Nguyen ◽

Michael Vigers ◽

Eric Sefah ◽

Susanna Seppala ◽

Jennifer Hoover ◽

...

Keyword(s):

Intrinsically Disordered ◽

C Terminus

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RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

Nucleic Acids Research ◽

10.1093/nar/gku700 ◽

2014 ◽

Vol 42 (15) ◽

pp. 10185-10195 ◽

Cited By ~ 4

Author(s):

Constanze Schelhorn ◽

James M.B. Gordon ◽

Lidia Ruiz ◽

Javier Alguacil ◽

Enrique Pedroso ◽

...

Keyword(s):

Rna Binding ◽

Titration Calorimetry ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Mobility Shift ◽

Cytoplasmic Polyadenylation ◽

C Terminus ◽

Self Association ◽

Mobility Shift Assay ◽

A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

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