Where is ccr5 synthesized in the cell

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Thus, the U CCR5 is the main coreceptor for R5 M-tropic viruses that are mainly isolated from patients in the early asymptomatic stage of HIV-infection. These chemokine receptors CCR5 and CXCR4 belong to the class of seven transmembrane G-protein coupled receptors and their natural ligands are key players in the recruitment of immune cells to sites of inflammation [ 5 , 6 ].

In addition, chemokine receptors, and especially CXCR4, are also implicated in several diseases, such as rheumatoid arthritis [ 7 , 8 ].

As AMD does not interact with any chemokine receptor other than CXCR4 and as the compound does not trigger any response by itself, it can be considered as a highly specific CXCR4 antagonist [ 26 — 28 ].

The availibity of stable and reliable in vitro models is a prerequisite for the successful setup of an accurate screening program for chemokine receptor antagonists. We compared these data with the expression of the receptors on U In addition, U CCR5 and U CXCR4 double-transfected cell line 3 panels, left column and in the single-transfected U CCR5 cell line 3 panels, central column and U CXCR4 cell line 3 panels, right column. Aspecific background fluorescence is indicated by the white peaks.

To examine if transfection yielded a fully functional surface receptor, we performed intracellular calcium mobilization assays on these double-transfected cells and compared their responses with those of the single-transfected cell lines.

To monitor chemokine-induced calcium responses, cells were loaded with the fluorescent calcium indicator Fluo-3 and fluorescence was measured after chemokine stimulation, using the FLIPR. CXCR4 cells upper panels and the double-transfected cells lower panels.

The double-transfected cells were stimulated by either chemokine at the same concentration. CD4 cells and in the double-transfected U CXCR4 cells. Then U The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells y-axis as function of time x-axis using the Fluorometric Imaging Plate Reader FLIPR.

Each data point represents the average value of the fluorescence measured in quadruplicate. The data of one representative experiment of four are shown. The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells y-axis as function of time x-axis using the FLIPR.

The data of one representative experiment out of four are shown. To investigate whether the U CCR5 and Jurkat. CCR5 cells. After 5 days of infection, a strong cytopathic effect was visible microscopically for NL4. CCR5 cells data not shown. On the other hand, the R5 strain BaL induced strong cytopathicity in U We also compared the infectability of the U AMD strongly inhibited the NL4.

CXCR4 cell line. The IC 50 value of the compound combination ratio against NL4. To determine the infectability of the U CXCR4 cells by HIV-1 more in detail, we investigated whether the cells could be infected with eight clinical isolates with distinct coreceptor usage i. The coreceptor phenotype of each of these viral isolates was determined using the single-transfected U The R5 isolates completely failed to infect the U After 5 days of infection, the virus-induced cytopathic effect was observed microscopically and the virus production was quantified in the supernatant using a viral p24 core Ag ELISA Figure 3.

Viral infection as assessed by both giant cell formation and p24 Ag production was detected with all clinical isolates used in this study. Only for CI 15 marginal residual p24 Ag production was detected in the presence of SCH-C, although no giant cell formation could be seen microscopically. Most importantly, for all isolates, no viral replication could be measured when U For CI 19 some minor p24 Ag production was detected, but again, no giant cell formation was visible microscopically.

The coreceptor use of each isolate is shown between brackets. The data of one representative experiment out of two are presented. However, the combination of both compounds completely inhibited giant cell formation by this dual-tropic HIV-1 isolate. Microscopic view of cytopathic effect giant cell formation at day 5 after infection of U Uninfected cells are shown as the negative control upper row. We have evaluated this new cell line for its sensitivity towards HIV-1 and chemokine-induced intracellular calcium mobilization and demonstrated its usefulness for the evaluation of new potential CCR5 and CXCR4 antagonists with potent antiviral activity.

We have selected the U CD4 cell line as the parental cell line because of its many advantages over other cell lines. Also, chemokine receptor-transfected U The apparent size is smaller than the theoretical molecular weight, but such discrepancy is not uncommon for GPCRs 38 , The incomplete denaturation by SDS alone probably resulted in a more compact shape of CCR5 and hence a faster migration The yield of soluble CCR5 was estimated to be approximately 0.

Taken together, these results establish that a sufficient amount of CCR5 polypeptide chains can be translated and solubilized in the cell-free reaction supplemented with Brij For all subsequent experiments, Brij was also included in the cell-free translation of CCR5 to improve its solubility unless otherwise stated. After performing the cell-free reaction in the presence of different surfactants, samples from the reaction mixtures were first centrifuged and supernatant protein fractions from each sample were then analyzed by dot blot, followed by spot densitometry analyses to compare the amounts of soluble CCR5.

The full names of each surfactant are presented in the Materials and Methods. Molecular chaperones such as GroEL-GroES are typically defined by their ability to assist the folding and assembly of proteins in a catalytic and non-consumptive manner During cell-free CCR5 synthesis, we took aliquots of the reaction mixture at various time points and halted protein synthesis by adding chloramphenicol.

At this instant, fully translated CCR5 was present in both unfolded and folded states. Half of the halted reaction mixture was subjected to pulse proteolysis to digest any unfolded protein.

We analyzed the undigested and digested samples by immunoblotting to monitor the translation reaction and the appearance of translated-folded CCR5 receptor, as shown in Fig.

The apparent rate constant for the folding of newly translated CCR5 polypeptides was estimated to be 8. The corresponding translation rate was approximately 5. The fit of the kinetic data to a simplified consecutive reaction model Materials and Methods is shown to describe the appearance of translated protein and the formation of translated-folded protein; this approach was used to estimate the apparent rate constants for these two processes. The rates of translation and folding of CCR5 were determined to be respectively 4.

Moreover, in the latter case, both the folding rate and the translation rate 3. Taken together, the results of kinetics of translation and folding for in vitro -translated CCR5 clearly suggest that GroEL-GroES plays an important role in CCR5 folding and can significantly increase the rate and efficiency of folding. Proteins in well-folded conformations usually show higher resistance to proteolysis than their unfolded counterparts The kinetics of proteolysis therefore reflects the folding status of the target protein.

Proteolysis appeared to comprise two digestion events that occurred on very different time scales. The fast digestion process was completed within a few minutes. The fast process can be attributed to unfolded CCR5 polypeptides that are highly susceptible to subtilisin proteolysis. The slow phase can be attributed to folded CCR5, which has a much higher resistance to digestion by subtilisin. The proteolysis curves could be fitted well with a two-exponential equation Fig. Although the digestion rate for the fast phase could be obtained from the exponential fit, yielding rate constants ranging from 2.

The proteolysis rate constant for the second phase was also obtained from curve fitting. As shown in Table 1 , the rate constants were 4.

Clearly, chaperones can facilitate the folding of CCR5 into a structure with a higher resistance to subtilisin. Figure 4 also shows that the relative amplitude of the slow phase is also distinct, i. The small relative amplitude for the case without chaperones suggests that the efficiency is very low when CCR5 folds without the assistance of chaperones, although it can fold spontaneously, whereas more folded CCR5 can be obtained in the presence of GroEL-GroES.

Control experiments were carried out with the addition of albumin. In these experiments, the proteolysis rate did not change noticeably, which suggested that the decreased proteolytic rate in the presence of chaperonin was not due to increased substrate concentration Fig.

Proteolytic reactions supplemented with bovine albumin were also performed as controls. The fit of the kinetic data to a 2-phase exponential equation is shown to describe the different CCR5 proteolytic processes.

To compare the folding status of CCR5 in the presence and absence of chaperonins, the ligand binding activity of CCR5 was measured. The binding interactions between the receptor and its ligand, eotaxin CCL11 44 , were evaluated using a quartz crystal microbalance QCM and the results are shown in Fig.

A typical time-course comprising association and dissociation phases is observed, indicating that the CCR5 receptors obtained are biologically active regardless of the addition of GroEL-GroES. The dissociation equilibrium constant K D , as assessed by fitting the kinetic data to a binding model 45 , was estimated to be 9. These values, which are independent of the total amounts of functional receptor analyzed, are in reasonable agreement with those measured previously, as well as those for another chemokine receptor, CCR3 45 , The results of our ligand binding measurements suggest that nascent CCR5 chains can spontaneously fold into their native state in the solubilizing agent Brij However, this process is very inefficient in the absence of GroEL-GroES, with a low folding rate and yield, as well as a reduction in binding affinity and structural stability.

All of these results suggest that the added chaperonin complex can promote the folding of newly translated CCR5. The fit of the kinetic data to a binding model is shown to obtain k a , k d and K D values. The chaperonin complex is unlikely to affect the rate or efficiency of transcription and translation. Instead, the increased production of soluble CCR5 is probable due to the more efficient folding and improved solubility of CCR5.

However, in some cases, GroEL alone can be sufficient to assist the folding of proteins without the cooperation of GroES 47 , We therefore also examined the effect of GroEL alone on the expression, folding kinetics, structural stability and biological activity of soluble CCR5 to assess the role of GroES in the folding of newly translated CCR5.

As shown in Supplementary Fig. Nevertheless, the addition of GroEL alone accelerated the formation of folded receptor Supplementary Fig. In terms of the structural stability and ligand-binding capacity of CCR5, the addition of GroEL alone to the synthesis process also exerted a noticeable effect Supplementary Fig.

The dissociation equilibrium constant also decreased from 9. However, the proteolytic rate 1. CCR5 polypeptides can be translated and solubilized in the cell-free system supplemented with Brij The interactions between surfactants and the chaperonins have been studied previously.

At high SDS concentrations, more than 0. It was also demonstrated by Goulhen et al. Brij, also known as n-dodecyl polyoxyethylene, is very similar in structure to n-octyl-polyoxyethylene. Given the previous studies it is believed that the non-ionic Brij surfactant used at a concentration of 0.

This process is considerably slower than the folding of normal single-domain proteins in cells Residual structures in the unfolded state are considered to be important for protein refolding and can make a substantial contribution to the faster refolding of a chemically denatured protein compared with its newly translated counterpart 17 , Given the initial folding state of the nascent CCR5 chain and the environmental dependency of protein folding 12 , 53 , the folding rate of CCR5 determined here is reasonable.

However, such a slow spontaneous folding process does not appear to be functionally beneficial and much faster folding is expected in vivo. The ligand binding measurements show that the receptor obtained in the absence of chaperones was biologically functional. This suggests that nascent CCR5 chains can spontaneously fold into their native state after being solubilized in Brij micelles, which parallels the structural adaptation of membrane proteins to the phospholipid bilayer in vivo 6.

Although the non-ionic surfactant Brij shows general advantages for the soluble cell-free expression of GPCR membrane proteins, its chemical structure and micellar aggregate, though not its amphiphilic character, are different from a bilayer. Folding in the phospholipid bilayer could therefore also be different. It was also observed that the processes of CCR5 proteolysis appeared to comprise two different enzymatic digestion events.

The rate of the faster digestion process is not affected by the addition of GroEL-GroES, but the slower phase is notably different. Given that the folded states are the same with and without the addition of GroEL-GroES, proteolysis would be expected to be the same for the slow phase. Without the assistance of chaperonins, the protein might not have fully folded into its native state or may have been present as a mixture of properly folded CCR5 and partially folded or misfolded states.

This observation is consistent with the ligand-binding results, where CCR5 produced without the addition of GroEL-GroES displayed a lower binding affinity, assuming that some folding intermediates or partially folded CCR5 also bind the ligand but with a weaker affinity than that of the native receptor.

The existence of the fast phase of proteolysis suggests that whether or not the chaperonin complex was added, unfolded CCR5 polypeptides were present in the cell-free protein synthesis system. However, the relative amplitude of the slow phase, which corresponds to the relative amount of folded CCR5, is noticeably different and is much greater in the presence of GroEL-GroES.

However, unfolded CCR5 was still present even with the addition of GroEL-GroES, which corresponds to the amplitude of the fast phase of proteolysis, although the relative amount was considerably decreased. Given that CCR5 can fold more efficiently in vivo , other chaperones could participate in the folding process. Although CCR5 can fold spontaneously, this process was slow and inefficient.

It is reasonable to expect that during the spontaneous folding of CCR5 in Brij micelles, the protein could become trapped in misfolded sates. However, with the assistance of GroEL-GroES, the folding free energy landscape becomes smoother, such that the protein is not trapped, resulting in a more rapid folding process Fig. Previous studies have highlighted the importance of hydrophobic surfaces of non-native substrate proteins in the recognition by GroEL 20 , The hydrophobic interaction between the cavity wall of GroEL and CCR5 polypeptide is also expected to play an important role in this process, as well as in affecting folding efficiency Taken together, the results suggest that GroEL-GroES can efficiently promote the functional folding of newly translated CCR5 by increasing the rate and efficiency of folding, as well as the final yield of soluble product.

We have also demonstrated that the folding of newly translated CCR5 can be promoted to some extent by the addition of GroEL alone. This result confirms the essential role of GroEL in mediating protein folding 22 , Given the crowded and complex interior environment of cells, which is inherently hostile to the productive folding of aggregation-prone proteins 55 , 56 , these results also raise the possibility that protein chaperones, in addition to natural lipids, can play an indispensable role in the folding of membrane protein on relevant timescales.

Besides, Tehver et al. This multi-timescale model was shown to agree well with experimental data; meanwhile, optimized chaperonin activity was shown to depend not only on the timescales in the reaction cycle of GroEL but also on the aggregation and folding characteristics of substrate proteins. The seven-transmembrane CCR5 is highly aggregation-prone and has distinct folding behaviors compared with water-soluble proteins, which probably affected chaperonin function significantly in this study.

In summary, we gained important new information about the folding of the membrane protein CCR5 and, in particular, the role of the GroEL-GroES chaperone system in this process. The His tag fused to CCR5 was used for protein detection or immobilization during protein function analysis.

Detergents or molecular chaperones were added to the cell-free reaction as required. After in vitro protein synthesis, samples of the reaction mixture were centrifuged and the supernatant protein fraction of each sample was analyzed by immunoblotting or SDS-PAGE to detect soluble CCR5.

The yield of soluble CCR5 in Brij was estimated by densitometric quantification after immunoblotting using a standard curve generated from a purified His-tagged GPCR of known concentration Supplementary Fig. All immunoblotting and fluorescence images were captured using an FLA imaging system Fujifilm. Detergents were added directly in the cell-free reaction at a concentration of 0. After protein synthesis, samples of the reaction mixture were centrifuged and the supernatant protein fraction of each sample was analyzed by dot blotting.

The relative amounts of soluble CCR5 in the presence of different detergents were then quantified by spot densitometry. The time-course for the appearance of newly translated, folded CCR5 receptors was measured using the method of Mallam and Jackson Half of this quenched translation reaction was immediately subjected to a 1-min pulse proteolysis by subtilisin Sigma-Aldrich at a concentration of 7.

We adopted a consecutive elementary reaction model. A, I and P represent the reactant, intermediate translated-unfolded protein and product folded protein , respectively. This greatly simplified approach to modeling the kinetic data makes it easier to estimate the rate constants to describe the appearance of full-length translated CCR5 and full-length folded receptor.

The rate equations were given by. The rate constant of translation can be obtained by fitting the kinetic data using Eq. The rate constant of folding can also be evaluated by substituting k trans into Eq. The fitted rate parameters can be found in Table 1.

CCR5 proteolysis was initiated by adding subtilisin directly to the reaction mixture after protein synthesis, at a final concentration of 5. The digested samples were analyzed by immunoblotting to determine the proteolytic susceptibility of CCR5 synthesized in the absence or presence of the added chaperonins. The concentrations of chaperonin used were the same as in the translation and folding kinetic study.

As controls, proteolytic reactions supplemented with bovine albumin were also performed. The final concentration of albumin was the same as that of the added chaperonins.