Effects of acidosis on the structure, composition, and function of adult murine femurs.

Physiologic pH is maintained in a narrow range through multiple systemic buffering systems. Metabolic Acidosis (MA) is an acid-base disorder clinically characterized by a decrease in systemic pH and bicarbonate (HCO3-) levels. Acidosis affects millions annually, resulting in decreased bone mineral density and bone volume and an increased rate of fracture. We developed an adult murine model of diet-induced metabolic acidosis via graded NH4Cl administration that successfully decreased systemic pH over a 14 day period to elucidate the effects of acidosis on the skeletal system. Blood gas analyses measured an increase in blood calcium and sodium levels indicating a skeletal response to 14 days of acidosis. MA also significantly decreased femur ultimate strength, likely due to modifications in bone morphology as determined from decreased microcomputed tomography values of centroid distance and area moment of inertia. These structural changes may be caused by aberrant remodeling based on histological data evidencing altered OCL activity in acidosis. Additionally, we found that acidosis significantly decreased bone CO3 content in a site-specific manner similar to the bone phenotype observed in human MA. We determined that MA decreased bone strength thus increasing fracture risk, which is likely caused by alterations in bone shape and compounded by changes in bone composition. Additionally, we suggest the temporal regulation of cell-mediated remodeling in MA is more complex than current literature suggests. We conclude that our model reliably induces MA and has deleterious effects on skeletal form and function, presenting similarly to the MA bone phenotype in humans.

Proteasome subunit α1 overexpression preferentially drives canonical proteasome biogenesis and enhances stress tolerance in yeast.

The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two ß-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.

An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes.

The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.

Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4.

Turnover of the 26S proteasome by autophagy is an evolutionarily conserved process that governs cellular proteolytic capacity and eliminates inactive particles. In most organisms, proteasomes are located in both the nucleus and cytoplasm. However, the specific autophagy routes for nuclear and cytoplasmic proteasomes are unclear. Here, we investigate the spatial control of autophagic proteasome turnover in budding yeast (Saccharomyces cerevisiae). We found that nitrogen starvation-induced proteasome autophagy is independent of known nucleophagy pathways but is compromised when nuclear protein export is blocked. Furthermore, via pharmacological tethering of proteasomes to chromatin or the plasma membrane, we provide evidence that nuclear proteasomes at least partially disassemble before autophagic turnover, whereas cytoplasmic proteasomes remain largely intact. A targeted screen of autophagy genes identified a requirement for the conserved sorting nexin Snx4 in the autophagic turnover of proteasomes and several other large multisubunit complexes. We demonstrate that Snx4 cooperates with sorting nexins Snx41 and Snx42 to mediate proteasome turnover and is required for the formation of cytoplasmic proteasome puncta that accumulate when autophagosome formation is blocked. Together, our results support distinct mechanistic paths in the turnover of nuclear versus cytoplasmic proteasomes and point to a critical role for Snx4 in cytoplasmic agglomeration of proteasomes en route to autophagic destruction.