Accumulation of Deleterious Passenger Mutations Is Associated with the Progression of Hepatocellular Carcinoma.

In hepatocellular carcinoma (HCC), somatic genome-wide DNA mutations are numerous, universal and heterogeneous. Some of these somatic mutations are drivers of the malignant process but the vast majority are passenger mutations. These passenger mutations can be deleterious to individual protein function but are tolerated by the cell or are offset by a survival advantage conferred by driver mutations. It is unknown if these somatic deleterious passenger mutations (DPMs) develop in the precancerous state of cirrhosis or if it is confined to HCC. Therefore, we studied four whole-exome sequencing datasets, including patients with non-cirrhotic liver (n = 12), cirrhosis without HCC (n = 6) and paired HCC with surrounding non-HCC liver (n = 74 paired samples), to identify DPMs. After filtering out putative germline mutations, we identified 187±22 DPMs per non-diseased tissue. DPMs number was associated with liver disease progressing to HCC, independent of the number of exonic mutations. Tumours contained significantly more DPMs compared to paired non-tumour tissue (258-293 per HCC exome). Cirrhosis- and HCC-associated DPMs do not occur predominantly in specific genes, chromosomes or biological pathways and the effect on tumour biology is presently unknown. Importantly, for the first time we have shown a significant increase in DPMs with HCC.

α-Hemoglobin-stabilizing protein (AHSP) perturbs the proximal heme pocket of oxy-α-hemoglobin and weakens the iron-oxygen bond.

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O2 bond, explaining previous reports that AHSP decreases αHb O2 affinity roughly 4-fold and promotes autooxidation due primarily to a 3-4-fold increase in the rate of O2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O2 dissociation rate and equilibrium constants, and reduced O2-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when ßHb is abundant but diverts αHb to a redox resistant holding state when ßHb is limiting.

Intrahepatic murine CD8 T-cell activation associates with a distinct phenotype leading to Bim-dependent death.

BACKGROUND & AIMS: Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo. METHODS: Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen. RESULTS: Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. CONCLUSIONS: This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.