The ocular microenvironment uses a poorly defined melanocortin 5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this new study, it was found that the MC5r-dependent regulatory immunity was an increase of CD11b+ F4/80+ Ly-6Clow Ly-6G+ CD39+ CD73+ APC in the spleen of post-EAU mice. These MC5r-dependent APC require adenosine 2A receptor (A2Ar) expression on T cells to activate EAU-suppressing CD25+ CD4+ FoxP3+ Treg cells. Therefore, in the recovery from autoimmune disease the ocular microenvironment induces tolerance through a melanocortin mediated expansion of Ly-6G+ regulatory APC in the spleen that utilize the adenosinergic pathway to promote activation of autoantigen-specific Treg cells.

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.mRNA degradation is a process involving the interaction and exchange of multiple multisubunit complexes and RNA binding proteins (8). Central to mRNA degradation is the removal of the poly(A) tail (deadenylation) that is controlled by a number of proteins associating with the mRNA in a structure termed the mRNP. Principal among these factors present in the mRNP are the poly(A) binding protein (PAB1), translation initiation and termination factors, the cytoplasmic deadenylases, and the factors that bind to the mRNA and elicit alterations in the mRNA degradative rate. The processes of mRNA degradation and deadenylation and the protein complexes that are involved are highly evolutionarily conserved from Saccharomyces cerevisiae to humans.The principal pathway for mRNA degradation in yeast proceeds through several steps. First, there is an initial trimming of about 15 to 20 nucleotides (nt) of the poly(A) tail to a length of about 60 to 80 nt that is specific for each mRNA and that appears to be carried out by PAN2/PAN3, presumably a cytoplasmic process (2,19,48). This trimming requires PAB1 and the translation termination factors eRF1 and eRF3 (5, 24), and all these factors are known to associate with each other (10, 23, 24, 29). Second, the major part of deadenylation utilizes the CCR4-NOT deadenylase complex (16,48). CCR4 is the catalytic component of this complex (7, 47) and shortens the poly(A) tail of mRNA to an end point size of about 8 to 12 nt (14). Poly(A) tail shortening down to an oligo(A) form (8 to 12 A's) may lead, in turn, to the reduced ability of PAB1 to bind the poly(A) tail that may alter the translation initiation...

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

Diabetes mellitus (DM) is a metabolic disease defined by elevated blood glucose (BG). DM is a global epidemic and the prevalence is anticipated to continue to increase. The ocular complications of DM negatively impact the quality of life and carry an extremely high economic burden. While systemic control of BG can slow the ocular complications they cannot stop them, especially if clinical symptoms are already present. With the advances in biodegradable polymers, implantable ocular devices can slowly release medication to stop, and in some cases reverse, diabetic complications in the eye. In this review we discuss the ocular complications associated with DM, the treatments available with a focus on localized treatments, and what promising treatments are on the horizon.

The evolutionarily conserved PUF proteins stimulate CCR4 mRNA deadenylation through binding to 3′ UTR sequences of specific mRNA. We have investigated the mechanisms by which PUF3 in Saccharomyces cerevisiae accelerates deadenylation of the COX17 mRNA. PUF3 was shown to affect PAN2 deadenylation of the COX17 mRNA independent of the presence of CCR4, suggesting that PUF3 acts though a general mechanism to affect deadenylation. Similarly, eIF4E, the cap-binding translation initiation factor, known to control CCR4 deadenylation, was shown to affect PAN2 activity in vivo. PUF3 was found to be required for eIF4E effects on COX17 deadenylation. Both eIF4E and PUF3 effects on deadenylation were shown, in turn, to necessitate a functional poly(A) binding protein (PAB1) in which removal of the RRM1 domain of PAB1 blocked both their effects on deadenylation. While removal of the proline-rich region (P domain) of PAB1 substantially reduces CCR4 deadenylation at non-PUF3 controlled mRNA and correspondingly blocked eIF4E effects on deadenylation, PUF3 essentially bypassed this P domain requirement. These results indicate that the PAB1-mRNP structure is critical for PUF3 action. We also found that multiple components of the CCR4-NOT deadenylase complex but not PAN2 interacted with PUF3. PUF3 appears, therefore, both to act independently of CCR4 activity, possibly through effects on PAB1-mRNP structure, and to be capable of retaining the CCR4-NOT complex.

Dependence on MC5r expression is with an APC that promotes or selectively activates IRBP-specific FoxP3+ TGF-β+ CD25+CD4+ Treg cells in the spleens of EAU-recovered mice.

The recovery of EAU, a mouse model of endogenous human autoimmune uveitis, is marked with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. This regulatory immunity is mediated by a melanocortin-driven suppressor APC that presents autoantigen and uses adenosine to activate an antigen-specific CD4(+) Tregs through the A2Ar. These cells are highly effective in suppressing uveitis, and they appear to be inducible Tregs. In this study, we determined whether they are inducible or natural Tregs and identified the dependent mechanism for the function of these post-EAU Tregs. The post-EAU spleen CD25(+)CD4(+) T cells were sorted for NRP-1 expression and transferred to recipient mice immunized for EAU. The sorted NRP-1(-), but not the NRP-1(+), Tregs suppressed EAU. These NRP-1(-) Tregs coexpress PD-1 and PD-L1. Treatment of naive APCs with α-MSH promoted a regulatory APC that induced CD25(+) CD4(+) Tregs in a CD73-dependent manner. These Tregs were PD-L1(+) PD-1(+) NRP-1(-) FOXP3(+) HELIOS(-) and suppressed EAU when transferred to recipient mice. In contrast, PD-1(-) T cells did not suppress EAU, indicating that PD-1 is necessary for the suppressive activity of iTregs. Moreover, these Tregs did not suppress effector T cells when the PD/-1/PD-L1 pathway was blocked. These results demonstrate that post-EAU Tregs are inducible Tregs, which use a PD-1/PD-L1 mechanism to suppress disease.

Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU), a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen, and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses, and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response, nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells, and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity, and the expression of MC5r and A2Ar on human immune cells, suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients.