Characterization of a novel binding partner of the melanocortin-4 receptor: attractin-like protein

The gene dosage effect of the MC4-R (melanocortin 4 receptor) on obesity suggests that regulation of MC4-R expression and function is critically important to the central control of energy homoeostasis. In order to identify putative MC4-R regulatory proteins, we performed a yeast two-hybrid screen of a mouse brain cDNA library using the mouse MC4-R intracellular tail (residues 303–332) as bait. We report here on one positive clone that shares 63% amino acid identity with the C-terminal part of the mouse attractin gene product, a single-transmembrane-domain protein characterized as being required for agouti signalling through the melanocortin 1 receptor. We confirmed a direct interaction between this ALP (attractin-like protein) and the C-terminus of the mouse MC4-R by glutathione S-transferase pulldown experiments, and mapped the regions involved in this interaction using N- and C-terminal truncation constructs; residues 303–313 in MC4-R and residues 1280–1317 in ALP are required for binding. ALP is highly expressed in brain, but also in heart, lung, kidney and liver. Furthermore, co-localization analyses in mice showed co-expression of ALP in cells expressing MC4-R in a number of regions known to be important in the regulation of energy homoeostasis by melanocortins, such as the paraventricular nucleus of hypothalamus and the dorsal motor nucleus of the vagus.

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Decreased Generation and Function of CD4+CD25hi T Regulatory Cells in Human STAT5b Deficiency.

Blood ◽

10.1182/blood.v106.11.768.768 ◽

2005 ◽

Vol 106 (11) ◽

pp. 768-768

Author(s):

Aileen Cleary ◽

Kari Nadeau ◽

Wenwei Tu ◽

Vivian Hwa ◽

Kira Y. Dionis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Dosage ◽

Foxp3 Expression ◽

Intermediate Phenotype ◽

Gamma Chain ◽

Gene Dosage Effect ◽

And Function

Abstract CD4+ CD25+ regulatory T cells (Tregs) are a well characterized population of cells that play an important role in limiting inflammation and in the maintenance of tolerance to self. Here we describe a patient with a homozygous missense mutation (A630P) in the STAT5b gene who clinically displays immune dysregulation in association with decreased numbers and function of Tregs. Freshly isolated or in vitro-derived CD4+CD25high Treg cells from this patient had low Foxp3 expression, did not suppress non Treg T-cell proliferation, and were unable to kill autologous CD4+CD25neg T cells compared to controls. CD25 expression in response to IL-2 did not increase on freshly isolated CD4 T cells and was decreased on T-cell blasts derived from the patient. The patients mother who was heterozygous for this mutation had an intermediate phenotype for all of these immune abnormalities, indicating a gene dosage effect. In contrast, IL-2 upregulated expression of the common gamma chain (γc) cytokine receptor and perforin by T cells normally. Activation-induced T-cell expression of CD40-ligand (CD154) and interferon-gamma (IFN-γ) were also normal in the patient. These results suggest that the STAT5 pathway propagates an important IL-2 mediated signal that is necessary for Treg generation and function in humans in vivo.

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Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter β-subunit

Biochemical Journal ◽

10.1042/bcj20161093 ◽

2017 ◽

Vol 474 (12) ◽

pp. 1981-1992 ◽

Cited By ~ 4

Author(s):

Whitney V. Christian ◽

Patricia M. Hinkle

Keyword(s):

Transmembrane Domain ◽

Basolateral Membrane ◽

Chimeric Proteins ◽

Transport Activity ◽

C Terminus ◽

Organic Solute Transporter ◽

Organic Solute ◽

And Function ◽

G Protein Coupled ◽

Solute Transporter

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostβ segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostβ were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostβ/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostβ TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostβ with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [3H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostβ is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex.

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Topology and Function of CcmD in Cytochrome c Maturation

Journal of Bacteriology ◽

10.1128/jb.00146-08 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3489-3493 ◽

Cited By ~ 18

Author(s):

Cynthia L. Richard-Fogal ◽

Elaine R. Frawley ◽

Robert G. Kranz

Keyword(s):

Escherichia Coli ◽

Cytochrome C ◽

Cytoplasmic Membrane ◽

Transmembrane Domain ◽

Cytochrome C Maturation ◽

Atp Binding Cassette Transporter ◽

C Terminus ◽

N Terminus ◽

Small Polypeptide ◽

And Function

ABSTRACT The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.

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Expression of Two Rye CENH3 Variants and Their Loading into Centromeres

Plants ◽

10.3390/plants10102043 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2043

Author(s):

Elena V. Evtushenko ◽

Evgeny A. Elisafenko ◽

Sima S. Gatzkaya ◽

Veit Schubert ◽

Andreas Houben ◽

...

Keyword(s):

Gene Dosage ◽

Central Component ◽

Gene Encoding ◽

Gene Dosage Effect ◽

Centromeric Chromatin ◽

Gene Copies ◽

Cereal Species ◽

Secale Cereale L ◽

And Function ◽

Mitosis And Meiosis

Gene duplication and the preservation of both copies during evolution is an intriguing evolutionary phenomenon. Their preservation is related to the function they perform. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the gene encoding this protein, while some (wheat, barley, rye) have two. Therefore, they represent a good model for a comparative study of the functional activity of the duplicated CENH3 genes and their protein products. We determined the organization of the CENH3 locus in rye (Secale cereale L.) and identified the functional motifs in the vicinity of the CENH3 genes. We compared the expression of these genes at different stages of plant development and the loading of their products, the CENH3 proteins, into nucleosomes during mitosis and meiosis. Using extended chromatin fibers, we revealed patterns of loading CENH3proteinsinto polynucleosomal domains in centromeric chromatin. Our results indicate no sign of neofunctionalization, subfunctionalization or specialization in the gene copies. The influence of negative selection on the coding part of the genes led them to preserve their conserved function. The advantage of having two functional genes appears as the gene-dosage effect.

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Evidence of a gene-dosage effect for the glucocorticoid receptor in trisomy 18 mice

Acta Endocrinologica ◽

10.1530/acta.0.114s095 ◽

1987 ◽

Vol 116 (3_Suppl) ◽

pp. S95-S96

Author(s):

D. VOGLIOLO ◽

H. WINKING ◽

R. KNUPPEN

Keyword(s):

Glucocorticoid Receptor ◽

Gene Dosage ◽

Dosage Effect ◽

Trisomy 18 ◽

Gene Dosage Effect

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BMP15 gene dosage as a relevant X-linked determinant of ovarian development and function

Endocrine Abstracts ◽

10.1530/endoabs.35.p646 ◽

2014 ◽

Author(s):

Raffaella Rossetti ◽

Irene Negri ◽

Chiara Castronovo ◽

Palma Finelli ◽

Luca Persani

Keyword(s):

Ovarian Development ◽

Gene Dosage ◽

And Function

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Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/137.2.407 ◽

1994 ◽

Vol 137 (2) ◽

pp. 407-422 ◽

Cited By ~ 8

Author(s):

E A Vallen ◽

W Ho ◽

M Winey ◽

M D Rose

Keyword(s):

Copy Number ◽

Gene Dosage ◽

Spindle Pole Body ◽

Direct Interaction ◽

Spindle Pole ◽

High Copy Number ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Recessive Mutations ◽

Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

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Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

Biochemical Journal ◽

10.1042/bj20020123 ◽

2002 ◽

Vol 364 (2) ◽

pp. 343-347 ◽

Cited By ~ 51

Author(s):

Gareth J.O. EVANS ◽

Alan MORGAN

Keyword(s):

Rat Brain ◽

Direct Interaction ◽

Secretory Vesicle ◽

Brain Membrane ◽

Synaptotagmin I ◽

Phosphorylated Form ◽

Order Of Magnitude ◽

And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

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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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