First, after their synthesis, DRiPs are rapidly targeted to the center of DALIS in maturing DCs. This rapid entry at a specific site implies that DALIS have a complex structural organization, probably involving specific scaffold proteins and enzymes likely to participate actively in improperly translated protein recruitment. Together with the fact that ribosomal proteins are found in close vicinity with DALIS (Lelouard et al., 2002
), this observation may also indicate that the translation apparatus and its associated chaperone machinery could be directly involved in the process of selection and inclusion of DRiPs into DALIS.
Second, DRiP ubiquitination occurs at this central location. This was primarily suggested by the rapid replacement of the GFP-Ub in DALIS monitored by FRAP, and by the appearance of high mol wt puromycin-labeled proteins forming ladders in the DALIS-enriched fractions after chasing the antibiotic for relatively short times. The detection in DALIS of the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E225K
, and the E3 ubiquitin ligase CHIP, required respectively for all the steps of misfolded protein ubiquitination, confirms that DALIS can serve as ubiquitination areas for DRiPs. Their enrichment in the central area of DALIS also suggests that aberrant protein entry and ubiquitination at specific sites are well coordinated. Moreover, although CHIP, which is likely to bind the ubiquitination substrates, remains tightly associated with DALIS under stringent conditions, the E1 and E225K
enzymes are efficiently removed by the same treatment, indicating that soluble components are also part of the DALIS machinery. This situation clearly distinguishes DALIS from classical protein aggregates generally defined as simple storage units of insoluble molecules (Kopito, 2000
). Importantly, the identified E1, E2, and E3 enzymes are all modulated during DC maturation; E1 and E2 being up-regulated while the insolubility of CHIP is increased, thus inferring that these enzymes have important function during DC maturation. The fact that ubiquitination occurs in DALIS also indicates that it is not a prerequisite for DRiPs targeting to DALIS, and may explain why other ubiquitinated proteasome substrates such as IkB-α are not incorporated in the aggregates (Lelouard et al., 2002
Third, DRiP accumulation in DALIS considerably extends the half-life of these abnormal proteins by preventing proteasome-mediated degradation. The efficiency in delaying DRiP degradation is quite high, as judged by the inability of proteasome inhibition to increase the quantity of puromycin-labeled proteins detected by immunoblot in the first 8 h after puromycin incorporation (16 h after the activation of DCs). The estimated half-time of residency of puromycin-labeled proteins (4 h after the puromycin pulse) and the stability of polyubiquitin levels in DALIS during the first 8 h also implies the existence of an equilibrium between the number of DRiPs entering and exiting DALIS after ubiquitination. Moreover, it suggests that at this stage of DC maturation (8–16 h of maturation), part of the ubiquitinated DRiPs can be stored and protected from degradation, even after exiting large DALIS structures (0.7 μm < diameter < 2.1 μm). In this condition, the stronger retention in DALIS of puromycin-labeled proteins observed after epoxomicin treatment could be due to an indirect effect of proteasome inhibition on many ubiquitin-based processes, rather than purely a block in the degradation of DRiPs stored in the aggregates.
Fourth, between 8 and 16 h of chase (16–24 h of DC maturation), DRiP degradation by the proteasome is clearly resumed.
DRiP fate during DC maturation has to be further integrated with the process of DALIS formation, maintenance, and disappearance. Videomicroscopy imaging using the GFP-Ub chimera indicates that DALIS are dynamic and can interact with each other (although in a heterogeneous manner). Therefore, DALIS formation could occur in several steps, first involving the assembly of “microaggregates,” followed by the fusion of the smaller and apparently more dynamic aggregates, and ending with the stabilization of the larger structures storing at that point the bulk of the DRiP production. The integrity of the larger structures could be dependent on the activity of the translation machinery, as demonstrated by the exquisite sensitivity of the aggregates to protein synthesis inhibitors. The translation machinery, in addition to producing DRiPs, could also provide some structural elements important to maintain the cohesion of large DALIS.
The efficient storage of large quantities of DRiPs during DC maturation is a puzzling phenomenon. The importance of DRiPs as an antigen source is such that a block/delay in their processing is likely to influence the MHC class I presentation in maturing DCs. The immunological consequences of this regulation remain to be fully evaluated. It has been recently proposed that DRiP processing and presentation is likely to be important for viral antigen presentation (Yewdell et al., 2003
); the storage of important viral determinants by DCs would therefore appear to be a counterproductive defense strategy. However, new evidences suggest that viral antigen presentation by DCs is occurring mainly via cross-presentation (Lizée et al., 2003
). This phenomenon is independent of direct viral protein synthesis by the DCs, and could be in fact favored by DALIS formation. Our work suggests that DALIS are organized structures performing coordinated biochemical functions, and that large protein aggregates might not be as amorphous as previously believed. Furthermore, the time frame of our observations implies that many important processes occurring in the first hours of DC activation have probably been overlooked.