Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.

Partial hepatectomy (PH) triggers hepatocyte proliferation–mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively.ConclusionPH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins.

Pregnancy is characterized by physiological adjustments in the maternal compartment. In this investigation, the influence of pregnancy on maternal liver was examined in CD-1 mice. Dramatic changes were observed in the size of the maternal liver during pregnancy. Livers doubled in weight from the nonpregnant state to day 18 of pregnancy. The pregnancy-induced hepatomegaly was a physiological event of liver growth confirmed by DNA content increase and detection of hepatocyte hyperplasia and hypertrophy. Growth of the liver was initiated following implantation and peaked at parturition. The expression and/or activities of key genes known to regulate liver regeneration, a phenomenon of liver growth compensatory to liver mass loss, were investigated. The results showed that pregnancy-dependent liver growth was associated with IL-6, TNF1α, NFkB, c-Jun, and IL-1β, but independent of HGF, FGF1, TNFR1, CAR, and PXR. Furthermore, maternal liver growth was associated with the activation of hepatic STAT3, β-catenin, and EGFR, but pregnancy did not activate hepatic c-Met. The findings suggest that the molecular mechanisms regulating pregnancy-induced liver growth and injury-induced liver regeneration exhibit overlapping features but are not identical. In summary, the liver of the mouse adapts to the demands of pregnancy via a dramatic growth response driven by hepatocyte proliferation and size increase. Remarkably, our findings provide a novel mouse model to investigate the regulation of hepatocyte proliferation, cell size increase, and liver growth.

Chronic kidney disease results in high serum urea concentrations leading to excessive protein carbamylation, primarily albumin. This is associated with increased cardiovascular disease and mortality. Multiple methods were used to address whether carbamylation alters albumin metabolism. Intravital two-photon imaging of the Munich Wistar Frömter (MWF) rat kidney and liver allowed us to characterize filtration and proximal tubule uptake and liver uptake. Microscale thermophoresis enabled quantification of cubilin (CUB7,8 domain) and FcRn binding. Finally, multiple biophysical methods including dynamic light scattering, small-angle X-ray scattering, LC-MS/MS and in silico analyses were used to identify the critical structural alterations and amino acid modifications of rat albumin. Carbamylation of albumin reduced binding to CUB7,8 and FcRn in a dose-dependent fashion. Carbamylation markedly increased vascular clearance of carbamylated rat serum albumin (cRSA) and altered distribution of cRSA in both the kidney and liver at 16 h post intravenous injection. By evaluating the time course of carbamylation and associated charge, size, shape, and binding parameters in combination with in silico analysis and mass spectrometry, the critical binding interaction impacting carbamylated albumin’s reduced FcRn binding was identified as K524. Carbamylation of RSA had no effect on glomerular filtration or proximal tubule uptake. These data indicate urea-mediated time-dependent carbamylation of albumin lysine K524 resulted in reduced binding to CUB7,8 and FcRn that contribute to altered albumin transport, leading to increased vascular clearance and increased liver and endothelial tissue accumulation.