共查询到20条相似文献,搜索用时 15 毫秒
1.
Michele E. Auldridge Hongnan Cao Saurabh Sen Laura P. Franz Craig A. Bingman Ragothaman M. Yennamalli George N. Phillips Jr. David Mead Eric J. Steinmetz 《PloS one》2015,10(4)
We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions. 相似文献
2.
Studies investigating the subcellular localization of periplasmic proteins have been hampered by problems with the export of green fluorescent protein (GFP). Here we show that a superfolding variant of GFP (sfGFP) is fluorescent following Sec-mediated transport and works best when the cotranslational branch of the pathway is employed. 相似文献
3.
Background
The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization.Methodology/Principal Findings
Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP.Conclusions/Significance
Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts. 相似文献4.
Shun Li Tatiana Flisikowska Mayuko Kurome Valeri Zakhartchenko Barbara Kessler Dieter Saur Alexander Kind Eckhard Wolf Krzysztof Flisikowski Angelika Schnieke 《PloS one》2014,9(7)
We are extending the Cre/loxP site-specific recombination system to pigs, focussing on conditional and tissue-specific expression of oncogenic mutations to model human cancers. Identifying the location, pattern and extent of Cre recombination in vivo is an important aspect of this technology. Here we report pigs with a dual fluorochrome cassette under the control of the strong CAG promoter that switches expression after Cre-recombination, from membrane-targeted tandem dimer Tomato to membrane-targeted green fluorescent protein. The reporter cassette was placed at the porcine ROSA26 locus by conventional gene targeting using primary mesenchymal stem cells, and animals generated by nuclear transfer. Gene targeting efficiency was high, and analysis of foetal organs and primary cells indicated that the reporter is highly expressed and functional. Cre reporter pigs will provide a multipurpose indicator of Cre recombinase activity, an important new tool for the rapidly expanding field of porcine genetic modification. 相似文献
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6.
Antonio Palmeri Gabriele Ausiello Fabrizio Ferrè Manuela Helmer-Citterich Pier Federico Gherardini 《Molecular & cellular proteomics : MCP》2014,13(9):2198-2212
Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area.Phosphorylation, the most widespread protein post-translational modification, is an important regulator of protein function. The addition of phosphate groups on serine, threonine, and tyrosine residues can modulate the activity of the target protein by inducing complex conformational changes, by modifying protein electrostatics, and by regulating domain-peptide interactions, as in 14-3-3 or SH2 domains, that specifically recognize phosphorylated residues. The standard experimental technique for the high-throughput identification of phosphorylation sites is mass spectrometry (1).Phosphorylation is catalyzed by protein kinases, a family that in humans comprises ∼540 members (2, 3). It is well understood that these enzymes recognize specific sequence motifs in their substrates (4, 5). Accordingly the sequence around the phosphorylation site is undisputedly the most important feature for phosphosite prediction (6, 7). However the “context,” in a broad sense, where these motifs occur is also important as sequence alone is not enough to achieve the observed specificity of phosphorylation. Therefore, several studies have characterized multiple aspects of phosphosites such as their preference for loops and disordered regions (reviewed in (8)), or the tendency of phosphoserines and phosphothreonines to occur in clusters (9), and these features have been used to improve the performance of phosphosite predictors (6, 7, 10–12). Moreover placing kinases and substrates in the context of protein interaction networks has been shown to improve the prediction of phosphorylation by specific kinases (13).Perhaps one of the most puzzling observations when looking at the phosphoproteome as a whole, is the fact that a large proportion of phosphorylation sites is poorly conserved. This has led to various hypotheses. First some sites may represent nonfunctional, possibly low-stoichiometry, phosphorylation events that are picked up because of the sensitivity of mass-spectrometry (14, 15). Indeed functionally characterized sites and those matching known kinase motifs are more conserved on average (15–17). However, although in biology function often equates with conservation, there could be genuinely functional fast-evolving phosphosites, that are responsible for species-specific differences in signaling and regulation. Moreover in some cases, especially in the regulation of protein-protein interactions, the exact position of the phosphosites may be unimportant (18, 19).Here we explore the issues of “context” and “conservation” of phosphorylation sites from the perspective of protein domains. To this end, we assembled a comprehensive database of phosphosites from publicly available sources and studied their proteome distribution with respect to the location and identity of protein domains. We focus on the human phosphoproteome because it has been very well characterized in a multitude of low- and high-throughput experiments, thus providing the opportunity for a comprehensive, proteome-wide, study. In particular, the issues we want to address are the following:
- Are specific domain types preferentially phosphorylated? Or conversely are some domains specifically depleted of phosphorylation sites?
- Can the domain context be used to improve the prediction of phosphorylation sites?
- What is the conservation pattern of phosphosites when looking at multiple instances of the same domain in the proteome?
7.
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10.
Thomas A. Roberts Francesca C. Norris Helen Carnaghan Dawn Savery Jack A. Wells Bernard Siow Peter J. Scambler Agostino Pierro Paolo De Coppi Simon Eaton Mark F. Lythgoe 《PloS one》2014,9(10)
Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. 相似文献
11.
Michael K. Wendt Joseph Molter Christopher A. Flask William P. Schiemann 《Journal of visualized experiments : JoVE》2011,(56)
Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis. 相似文献
12.
《Fly》2013,7(6):340-342
Green fluorescent protein-based reporters are commonly used to investigate protein nucleocytoplasmic transport. In this study we developed a novel reporter GFP2-GST which consists of 2 copies of GFP and 1 copy of GST, and tested it in two commonly used Drosophila cell lines. The size of the GFP2-GST reporter exceeds the passive diffusion limit across the nuclear pore complexes. It shows an exclusive cytoplasmic localization and displays a restrictive nuclear localization when a nuclear localization signal is appended. This reporter will largely facilitate the characterization and identification of NLS sequences in the fly proteome. 相似文献
13.
The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.Download video file.(37M, mov) 相似文献
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16.
PDBsum1 is a standalone set of programs to perform the same structural analyses as provided by the PDBsum web server (https://www.ebi.ac.uk/pdbsum). The server has pages for every entry in the Protein Data Bank (PDB) and can also process user‐uploaded PDB files, returning a password‐protected set of pages that are retained for around 3 months. The standalone version described here allows for in‐house processing and indefinite retention of the results. All data files and images are pre‐generated, rather than on‐the‐fly as in the web version, so can be easily accessed. The program runs on Linux, Windows, and mac operating systems and is freely available for academic use at https://www.ebi.ac.uk/thornton-srv/software/PDBsum1. 相似文献
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18.
Shaoliang Hu Mingrong Wang Guoping Cai Mingyue He 《The Journal of biological chemistry》2013,288(43):30855-30861
Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its “codon-preferred” DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ∼2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10−8
m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids. 相似文献
19.
YongQiang Wang Mingxiang Liao Nicholas Hoe Poulomi Acharya Changhui Deng Andrew N. Krutchinsky Maria Almira Correia 《The Journal of biological chemistry》2009,284(9):5671-5684
Cytochromes P450 (P450s) incur phosphorylation. Although the precise role
of this post-translational modification is unclear, marking P450s for
degradation is plausible. Indeed, we have found that after structural
inactivation, CYP3A4, the major human liver P450, and its rat orthologs are
phosphorylated during their ubiquitin-dependent proteasomal degradation.
Peptide mapping coupled with mass spectrometric analyses of CYP3A4
phosphorylated in vitro by protein kinase C (PKC) previously
identified two target sites, Thr264 and Ser420. We now
document that liver cytosolic kinases additionally target Ser478 as
a major site. To determine whether such phosphorylation is relevant to in
vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or
triple Ala mutations of these residues were heterologously expressed in
Saccharomyces cerevisiae pep4Δ strains. We found that relative
to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and
this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and
S478A/T264A/S420A double and triple mutants. Similar relative
S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To
determine whether phosphorylation enhances CYP3A4 degradation by enhancing its
ubiquitination, CYP3A4 ubiquitination was examined in an in vitro
UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase
A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation.
cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but
not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system.
Together, these findings indicate that phosphorylation of CYP3A4
Ser478, Thr264, and Ser420 residues by
cytosolic kinases is important both for its ubiquitination and proteasomal
degradation and suggest a direct link between P450 phosphorylation,
ubiquitination, and degradation.Hepatic cytochromes P450
(P450s)3 are integral
endoplasmic reticulum (ER)-anchored hemoproteins engaged in the oxidative
biotransformation of various endo- and xenobiotics. Of these, human CYP3A4 is
the most dominant liver enzyme, accounting for >30% of the hepatic
microsomal P450 complement, and responsible for the oxidative metabolism of
over 50% of clinically relevant drugs
(1). In common with all the
other ER-bound P450s, CYP3A4 is a monotopic protein with its N-terminal
≈33-residue domain embedded in the ER membrane with the bulk of its
structure in the cytosol. Our in vivo studies of the heterologously
expressed CYP3A4 in the yeast Saccharomyces cerevisiae as well as of
its rat liver CYP3A2/3A23 orthologs in primary hepatocytes have revealed that
human and rat liver CYPs 3A are turned over via ubiquitin (Ub)-dependent
proteasomal degradation (UPD)
(2–8).
Thus, CYPs 3A represent excellent prototypic substrates of ER-associated
degradation (ERAD), specifically of the ERAD-C pathway
(6–11).
Consistent with this CYP3A ERAD process, our studies of in vivo
and/or in vitro reconstituted systems have led us to conclude that
CYPs 3A are ubiquitinated by the UBC7/gp78 Ub-ligase complex and recruited by
the p97-Npl4-Ufd1 complex before their degradation by the 26 S proteasome
(4–8,
12). Because all these
processes are energy-dependent, it is not surprising that in vitro
reconstitution of CYP3A4 UPD requires ATP. However, inclusion of
γ-S-[32P]ATP in an in vitro reconstituted
CYP3A4 ubiquitination system catalyzed by rat liver cytosolic fraction II
(FII) resulted in CYP3A4 protein phosphorylation, i.e.
γ-[32P]phosphoryl transfer onto CYP3A4 target residues
(13,
14). This phosphorylation was
enhanced after cumene hydroperoxide (CuOOH)-mediated CYP3A4 inactivation. The
physiological role, if any, of this CYP3A4 post-translational modification is
unclear.CYP3A4 is not the only P450 that is phosphorylated. Since the in
vitro phosphorylation of a hepatic P450 (CYP2B4) by cAMP-dependent
protein kinase A (PKA) was first described
(15), various P450s,
particularly those belonging to the subfamily 2, were documented to be
phosphorylated in cell-free systems, hepatocyte incubations, and intact
animals
(16–32).
Common features of such P450 phosphorylation were the presence of a
cytosolically exposed PKA recognition sequence (RRXS) with the Ser
residue as the exclusive kinase target, and the ensuing loss of prosthetic
heme, conversion to the inactive P420 species, and consequent dramatic
functional inactivation
(15–20).
Studies in intact rats also identified CYPs 3A and 2C6 as kinase targets
(21). Although both these
P450s lack the hallmark PKA recognition sequence, apparently they possess
secondary PKA targeting sequences or are phosphorylated by other protein
kinases such as PKC. Indeed, in vitro studies revealed that P450s
were phosphorylated in an isoform-dependent manner by either PKA or PKC,
except for CYP2B1, which was heavily phosphorylated by both
(20). Over the years since
this particular post-translational P450 modification was recognized, it has
been assigned various functional roles
(17,
29–33).
Among these, as first proposed by Taniguchi et al.
(16) and later explored both
by Eliasson et al.
(23–26)
and us (13,
14), P450 phosphorylation
served as a marker for its degradation. Accordingly, the phosphorylation of
CYP2E1Ser129 and CYP3A1Ser393 by a microsomal
cAMP-dependent protein kinase has been proposed to predispose these P450s but
not the similarly phosphorylated CYP2B1 to proteolytic degradation by an
integral ER Mg2+-ATP-activated serine protease
(23–27).
However, heterologous expression of CYP2E1S129A/S129G site-directed mutants in
COS7 cells apparently had no effect on its relative stability thereby
revealing that if CYP2E1 phosphorylation is important for its degradation
(34,
35), then alternate Ser/Thr
residues (i.e. in plausible secondary PKA recognition sites,
Lys-Lys-Ser209-Lys and Lys-Lys-Ser449-Ala) may be
recruited.On the other hand, on the basis of rapid phosphorylation of
CuOOH-inactivated CYP3A4 that precedes its ubiquitination and 26 S proteasomal
degradation in an in vitro liver cytosolic FII-catalyzed system, we
have proposed that CYP3A4 phosphorylation was essential for targeting it to
proteins participating in its UPD/ERAD
(13). Indeed, several examples
of similar phosphorylation for targeting proteins to UPD exist, of which
IκBα phosphorylation is the most notable and perhaps the best
documented
(36–47;
see “Discussion”).Our in vitro studies with specific kinase inhibitors as probes
identified both PKC and PKA as the major FII kinases responsible for CYP3A4
phosphorylation (14). Indeed,
in vitro model studies of CYP3A4 with PKC as the kinase, coupled with
lysylendopeptidase C (Lys-C) digestion of the phosphorylated protein and
liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of the
Lys-C digests, identified two PKC-phosphorylated CYP3A4 peptides
258ESRLEDpTQK266 and
414FLPERFpSK421 unambiguously phosphorylated at
Thr264 and Ser420
(14). These same residues were
also phosphorylated in corresponding studies with
PKA.4 Furthermore,
although both native and CuOOH-inactivated CYP3A4 were phosphorylated at
Thr264, Ser420 phosphorylation was particularly enhanced
after CuOOH-mediated CYP3A4 inactivation
(14). Corresponding studies of
CuOOH-inactivated CYP3A4 using rat liver cytosolic FII as the source of the
kinase(s), revealed 32P phosphorylation of both these peptides as
well as that of an additional CYP3A4 peptide
477LS(p)LGGLLQPEKPVVLK492. Unlike the unambiguous mass
spectrometric identification of Thr264 and Ser420 as the
phosphorylated CYP3A4 residues, the phosphorylation of Ser478, the
only plausible phosphorylatable residue in this 32P-labeled
peptide, was not similarly established. Nevertheless, the predominant
phosphorylation of Thr264 in native CYP3A4
(14), but of two additional
residues in the CuOOH-inactivated enzyme, is consistent with the
inactivation-induced structural unraveling of this enzyme with exposure of
otherwise concealed and/or kinase-inaccessible domains
(48). Such unraveling of
CYP3A4 protein stems from the irreversible modification of its active site by
fragments generated from CuOOH-mediated oxidative destruction of its
prosthetic heme (49). In this
study, using mass spectrometric analyses of Lys-C digests of
FII-phosphorylated CYP3A4, we have provided unambiguous evidence that in
addition to Thr264 and Ser420, Ser478 is
indeed phosphorylated. More importantly, through alanine-scanning mutagenesis
of these three residues, we now document that although neither the structural
conformation nor the catalytic function of this triple CYP3A4T264A/S420A/S478A
mutant is altered, its degradation after heterologous expression in S.
cerevisiae is significantly impaired. This is also true of
CYP3A4T264A/S420A/S478A mutant degradation in human embryonic kidney (HEK293T)
cells. Furthermore, using an in vitro reconstituted CYP3A4
ubiquitination system, catalyzed by human Ub-conjugating E2 enzyme UBC7 and
integral ER protein gp78 as the E3 Ub ligase
(12), we document that
PKA/PKC-mediated phosphorylation of the wild type CYP3A4 (CYP3A4wt)
considerably enhanced its UBC7/gp78-mediated ubiquitination. Together these
findings reveal the critical importance of CYP3A4 phosphorylation at these
residues for its UPD and suggest a direct link between phosphorylation and its
ubiquitination and degradation. 相似文献