Assembly pathway of the Mammalian proteasome base subcomplex is mediated by multiple specific chaperones.

The 26S proteasome is an enzymatic complex that degrades ubiquitinated proteins in eukaryotic cells. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The latter is further divided into the lid and base subcomplexes. While the mechanism involved in the assembly of the CP is well investigated, that of the RP is poorly understood. Here, we show that the formation of the mammalian base subcomplex involves three distinct modules, where specific pairs of ATPase subunits are associated with the distinct chaperones p28, S5b, or p27. The process of base formation starts from association of the p28-Rpt3-Rpt6-Rpn14 complex with the S5b-Rpt1-Rpt2-Rpn1 complex, followed by incorporation of the p27-Rpt5-Rpt4 complex and Rpn2, where p28, S5b, and p27 regulate the associations between the modules. These chaperones dissociate before completion of 26S proteasome formation. Our results demonstrate that base assembly is facilitated by multiple proteasome-dedicated chaperones, like CP assembly.

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome.

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

The solute carrier family 15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice.

BACKGROUND & AIMS: Solute carrier family 15 (SLC15) A4 is a proton-coupled histidine and oligopeptide cotransporter expressed by the immune and nervous systems and associated with disorders such as inflammatory bowel diseases and systemic lupus erythematosus. High levels of SLC15A4 transcripts were observed in human antigen-presenting cells, including dendritic cells, activated macrophages, and B cells. However, the roles of SLC15A4 in the immune regulation are not known. We investigated the function of SLC15A4 in the innate immune system. METHODS: We created SLC15A4-deficient (SLC15A4(-/-)) mice and compared Toll-like receptor 9 and NOD1-dependent innate immune responses between SLC15A4(-/-) and control (SLC15A4(+/+)) mice. RESULTS: SLC15A4 deficiency impaired CpG-induced production of interleukin-12, interleukin-15, and interleukin-18 by dendritic cells. Correspondingly, SLC15A4(-/-) mice developed a less severe form of Th1-dependent colitis than SLC15A4(+/+) mice. Increased lysosomal histidine, in the absence of SLC15A4, appears to negatively regulate Toll-like receptor 9 function by inhibiting the proteolytic activities of cathepsins B and L. SLC15A4(-/-) mice also had a severe defect in NOD1-dependent cytokine production, indicating that SLC15A4 functions as a transporter of the NOD1 ligand. CONCLUSIONS: SLC15A4 promotes colitis through Toll-like receptor 9 and NOD1-dependent innate immune responses. Histidine homeostasis within intracellular compartments is important for eliciting effective innate immune responses.