Retinol and Retinol-Binding Protein Stabilize Transthyretin <i>via</i> Formation of Retinol Transport Complex

Hyung, Suk Joon; Deroo, Stéphanie; Robinson, Carol V.

doi:10.1021/cb100144v

Cited by 47 publications

(56 citation statements)

References 33 publications

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“…The dynamics of the L7/L12 proteins were investigated using an MS strategy that has proven successful to elucidate the subunit exchange dynamics of other protein complexes (30)(31)(32)(33). Specifically, ribosomes are uniformly labeled with stable isotopes ( 13 C and 15 N) to provide sufficient mass differences to resolve heterocomplexes formed with wild-type ribosomes.…”

Section: Isotopically Labeled Ribosomes Maintain Interactionsmentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. Based on kinetic analysis we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.Protein biosynthesis is carried out by the ribosomal machinery and requires interaction of several translation factors with the ribosomal stalk complex. In bacteria the base of the stalk region, interacting with the rRNA of the 50S large ribosomal subunit, is composed of adjacent proteins L11 and L10 (1). The C-terminal domain (CTD) of L10 is a long and mobile helix which interacts with two dimers of protein L12 (2). Three pairs of L12 are observed in thermophilic bacteria and archaea (3-6) and L12 is the only protein present as multiple copies on the ribosome. The N-terminal domain (NTD) of L12 is responsible for dimerization and for anchoring of the protein to the ribosome (7). In Escherichia coli (E. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts coli), L12 is partially N-acetylated to form protein L7 (8). The ratio of L7 to L12 is known to change throughout the growth phase (8-10) and has been linked to variation in the stability of the stalk complex via hydrogen exchange mass spectrometry (MS) experiments (11). L12 CTD and NTD are connected via a flexible hinge region providing mobility to the highly structured CTD (12, 13). These highly mobile proteins have eluded high resolution X-ray analysis for many years. Only recently the atomic structure of a truncated stalk complex of a thermophilic bacterium revealed the binding mode of the NTD of L12 proteins to L10 (4). The tight antiparallel binding of the L12 NTDs is similar to that observed by NMR for the E. coli L7/L12 dimer in solution (14). The mobility and dynamics o...

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Section: Isotopically Labeled Ribosomes Maintain Interactionsmentioning

confidence: 99%

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

et al. 2012

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“…Additionally, the rapid analysis possible with ESI-MS makes it convenient for following subunit exchange in real time [38]. Despite these benefits, only a small number of studies applying ESI-MS to multimeric protein subunit exchange have been reported [39][40][41][42][43][44][45][46][47][48].…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason

Jergic

et al. 2013

J. Am. Soc. Mass Spectrom.

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Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions AbstractSingle-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry. Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNAbinding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange.Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

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“…Expansion of these studies to larger group numbers would confirm our findings and further demonstrate the utility of RBP4 as a biomarker of ATTRm cardiac amyloid disease. Moreover, this study did not include examination of serum vitamin A levels, potentially important as retinol is a key player in the recognition and binding of RBP4 to TTR [4,21,41]. Unfortunately, accurate measurement of vitamin A is challenging as levels are homeostatically controlled (50-200 g/dL) [42] and serum retinol concentrations are only an indirect reflection of total vitamin A in the liver.…”

Section: Discussionmentioning

confidence: 99%

“…RBP4-TTR complexes are formed in the ER [1,2] and their release from the liver into circulation is dependent on retinol binding to RBP4 [3]. Consequently, 95% of circulating RBP4 is bound to TTR [4,5]. The serum concentration is normally 42 µg/mL, but elevated levels have been described in type 2 diabetes [6], obesity [7], and inflammatory cardiomyopathy [8].…”

Section: Introductionmentioning

confidence: 99%

Retinol-binding protein 4 in transthyretin-associated forms of cardiac amyloidosis: differences in the pathobiologies of mutant (ATTRm) and wild-type (ATTRwt) diseases

Koch¹,

Chiswick²,

Cui³

et al. 2017

Clin Diagn Pathol

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The role of retinol-binding protein 4 (RBP4), a natural binding partner of plasma circulating transthyretin (TTR), in TTR-associated cardiomyopathies is unknown. RBP4 is a small (21 kDa) protein that normally functions as a transporter for all-trans retinol (Vitamin A) and travels in the bloodstream as a ternary complex bound to TTR. Previous in vitro studies have demonstrated that RBP4 stabilizes native (tetrameric) TTR and prevents its disassembly into monomers, the key step in TTR amyloid fibril formation. Though increased serum concentrations of RBP4 have been reported in non-amyloidotic cardiomyopathies, there is little information about circulating levels in the inherited (ATTRm) and acquired (ATTRwt) forms of TTR-associated cardiac amyloid disease. Our study objectives were to investigate TTR amyloid-infiltrated cardiac tissue for the presence of RBP4 and to compare serum levels of the protein in ATTRm, ATTRwt and controls. We hypothesized that there would be histological and serological differences in RBP4 between ATTR patient and control samples. In the present study, we demonstrate that RBP4 is highly abundant in ATTRm cardiac tissue surrounding amyloid-infiltrated regions and, to a lesser extent, in ATTRwt specimens. Serum levels of RBP4 are significantly lower in ATTRm compared to ATTRwt (p < 0.0001) and healthy controls (p < 0.0011), with significant correlation between circulating RBP4 concentration and cardiac troponin-I (c-TnI) in our ATTRwt cohort. These data suggest, for the first time, that the pathobiologies of ATTRm and ATTRwt are dissimilar, and provide support for RBP4 as a serum biomarker of amyloid cardiomyopathy in ATTRm.

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Retinol and Retinol-Binding Protein Stabilize Transthyretin via Formation of Retinol Transport Complex

Cited by 47 publications

References 33 publications

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Retinol-binding protein 4 in transthyretin-associated forms of cardiac amyloidosis: differences in the pathobiologies of mutant (ATTRm) and wild-type (ATTRwt) diseases

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