A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with ten-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable -4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.

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Plasma/serum proteomics: depletion strategies for reducing high-abundance proteins for biomarker discovery

Bioanalysis ◽

10.4155/bio-2019-0145 ◽

2019 ◽

Vol 11 (19) ◽

pp. 1799-1812 ◽

Cited By ~ 6

Author(s):

Pey Yee Lee ◽

Junaida Osman ◽

Teck Yew Low ◽

Rahman Jamal

Keyword(s):

Biomarker Discovery ◽

Dynamic Range ◽

High Abundance ◽

Future Perspectives ◽

Proteomics Analysis ◽

Wide Dynamic Range ◽

Serum Proteomics ◽

Detection And Identification ◽

Potential Biomarkers ◽

Abundance Proteins

Plasma and serum are widely used for proteomics-based biomarker discovery. However, analysis of these biofluids is highly challenging due to the complexity and wide dynamic range of their proteomes. Notably, highly abundant proteins tend to obscure the detection of potential biomarkers that are usually of lower concentrations. Among the strategies to resolve this problem are: depletion of high-abundance proteins, enrichment of low abundant proteins of interest and prefractionation. In this review, we focus on current and emerging depletion techniques used to enhance the detection and identification of the less abundant proteins in plasma and serum. We discuss the applications and contributions of these methods to proteomics analysis of plasma and serum alongside their limitations and future perspectives.

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A baton-relay mechanism for ER retrieval signal capture and proofreading by the KDEL receptor

10.1101/2021.03.21.436307 ◽

2021 ◽

Author(s):

Andreas Gerondopoulos ◽

Philipp Bräuer ◽

Tomoaki Sobajima ◽

Zhiyu Wu ◽

Joanne L Parker ◽

...

Keyword(s):

Molecular Basis ◽

Carboxyl Terminus ◽

Acidic Ph ◽

Receptor Affinity ◽

Neutral Ph ◽

Binding Cavity ◽

Arginine Residues ◽

Ph Dependent ◽

Kdel Receptor ◽

Initial Capture

The KDEL-retrieval pathway captures escaped ER proteins with a KDEL or variant C-terminal signal at acidic pH in the Golgi and releases them at neutral pH in the ER. To address the mechanism of signal binding and the molecular basis for differences in signal affinity, we determined the HDEL and RDEL bound structures of the KDEL-receptor. Affinity differences are explained by interactions between the variable -4 position of the signal and W120, whereas initial capture of retrieval signals by their carboxyl-terminus is mediated by a baton-relay mechanism involving a series of conserved arginine residues in the receptor. This explains how the signal is first captured and then pulled into the binding cavity. During capture, retrieval signals undergo a selective proofreading step involving two gatekeeper residues D50 and E117 in the receptor. These mechanisms operate upstream of the pH-dependent closure of the receptor and explain the selectivity of the KDEL-retrieval pathway.

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Depletion of High-Abundance Proteins in Plasma by Immunoaffinity Subtraction for Two-Dimensional Difference Gel Electrophoresis Analysis

Cardiovascular Proteomics ◽

10.1385/1-59745-214-9:351 ◽

2006 ◽

pp. 351-364 ◽

Cited By ~ 1

Author(s):

Verónica M. Dardé ◽

Maria G. Barderas ◽

Fernando Vivanco

Keyword(s):

Gel Electrophoresis ◽

High Abundance ◽

Two Dimensional ◽

Difference Gel Electrophoresis ◽

Abundance Proteins

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High Abundance Proteins: Proteomer’s Thorns in the Flesh?

Journal of Proteomics & Bioinformatics ◽

10.4172/jpb.1000e35 ◽

2017 ◽

Vol 10 (7) ◽

Author(s):

Ravi Gupta

Keyword(s):

High Abundance ◽

Abundance Proteins

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Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid

Biotechnology and Applied Biochemistry ◽

10.1042/ba20080015 ◽

2009 ◽

Vol 52 (2) ◽

pp. 159 ◽

Cited By ~ 25

Author(s):

Margareta Ramström ◽

Aida Zuberovic ◽

Caroline Grönwall ◽

Jörg Hanrieder ◽

Jonas Bergquist ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

High Abundance ◽

Abundance Proteins

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Unique functional properties of a member of the Fushi Tarazu-Factor 1 family from Schistosoma mansoni

Biochemical Journal ◽

10.1042/bj20040489 ◽

2004 ◽

Vol 382 (1) ◽

pp. 337-351 ◽

Cited By ~ 10

Author(s):

Benjamin BERTIN ◽

Souphatta SASORITH ◽

Stéphanie CABY ◽

Frédérik OGER ◽

Jocelyne CORNETTE ◽

...

Keyword(s):

Amino Acids ◽

Ligand Binding ◽

Schistosoma Mansoni ◽

Transcriptional Activity ◽

Binding Pocket ◽

Activation Function ◽

Orphan Receptor ◽

Fushi Tarazu ◽

Dependent Activity ◽

Arginine Residues

SmFtz-F1 (Schistosoma mansoni Fushi Tarazu-Factor 1) belongs to the Ftz-F1 subfamily of nuclear receptors, but displays marked structural differences compared with its mammalian homologues SF-1 (steroidogenic factor-1) or liver receptor homologue-1. These include a long F domain (104 amino acids), an unusually large hinge region (133 amino acids) and a poorly conserved E-domain. Here, using Gal4 constructs and a mammalian two-hybrid assay, we have characterized the roles of these specific regions both in the transcriptional activity of the receptor and in its interactions with cofactors. Our results have shown that, although the AF-2 (activation function-2) region is the major activation function of the receptor, both the F and D domains are essential for AF-2-dependent activity. Modelling of SmFtz-F1 LBD (ligand-binding domain) and structure-guided mutagenesis allowed us to show the important role of helix H1 in maintaining the structural conformation of the LBD, and suggested that its autonomous transactivation activity, also observed with SF-1, is fortuitous. This strategy also allowed us to study an eventual ligand-dependence for this orphan receptor, the predicted three-dimensional models suggesting that the SmFtz-F1 LBD contains a large and well-defined ligand-binding pocket sealed by two arginine residues orientated towards the interior of the cavity. Mutation of these two residues provoked a loss of transcriptional activity of the receptor, and strongly reduced its interaction with SRC1 (steroid receptor cofactor-1), suggesting a ligand-dependent activity for SmFtz-F1. Taken together, our results argue for original and specific functional activities for this platyhelminth nuclear receptor.

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