Hepatic protein kinase Cbeta deficiency mitigates late-onset obesity.

Although aging is associated with progressive adiposity and a decline in liver function, the underlying molecular mechanisms and metabolic interplay are incompletely understood. Here, we demonstrate that aging induces hepatic protein kinase Cbeta (PKCß) expression, while hepatocyte PKCß deficiency (PKCßHep-/-) in mice significantly attenuates obesity in aged mice fed a high-fat diet. Compared with control PKCßfl/fl mice, PKCßHep-/- mice showed elevated energy expenditure with augmentation of oxygen consumption and carbon dioxide production which was dependent on ß3-adrenergic receptor signaling, thereby favoring negative energy balance. This effect was accompanied by induction of thermogenic genes in brown adipose tissue (BAT) and increased BAT respiratory capacity, as well as a shift to oxidative muscle fiber type with an improved mitochondrial function, thereby enhancing oxidative capacity of thermogenic tissues. Furthermore, in PKCßHep-/- mice, we determined that PKCß overexpression in the liver mitigated elevated expression of thermogenic genes in BAT. In conclusion, our study thus establishes hepatocyte PKCß induction as a critical component of pathophysiological energy metabolism by promoting progressive hepatic and extrahepatic metabolic derangements in energy homeostasis, contributing to late-onset obesity. These findings have potential implications for augmenting thermogenesis as a means of combating aging-induced obesity.

hsp70-dependent antiviral immunity against cytopathic neuronal infection by vesicular stomatitis virus.

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-ß in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.

hsp70 and a novel axis of type I interferon-dependent antiviral immunity in the measles virus-infected brain.

The major inducible 70-kDa heat shock protein (hsp70) is host protective in a mouse model of measles virus (MeV) brain infection. Transgenic constitutive expression of hsp70 in neurons, the primary target of MeV infection, abrogates neurovirulence in neonatal H-2(d) congenic C57BL/6 mice. A significant level of protection is retained after depletion of T lymphocytes, implicating innate immune mechanisms. The focus of the present work was to elucidate the basis for hsp70-dependent innate immunity using this model. Transcriptome analysis of brains from transgenic (TG) and nontransgenic (NT) mice 5 days after infection identified type I interferon (IFN) signaling, macrophage activation, and antigen presentation as the main differences linked to survival. The pivotal role of type I IFN in hsp70-mediated protection was demonstrated in mice with a genetically disrupted type I IFN receptor (IFNAR(-/-)), where IFNAR(-/-) eliminated the difference in survival between TG and NT mice. Brain macrophages, not neurons, are the predominant source of type I IFN in the virus-infected brain, and in vitro studies provided a mechanistic basis by which MeV-infected neurons can induce IFN-ß in uninfected microglia in an hsp70-dependent manner. MeV infection induced extracellular release of hsp70 from mouse neuronal cells that constitutively express hsp70, and extracellular hsp70 induced IFN-ß transcription in mouse microglial cells through Toll-like receptors 2 and 4. Collectively, our results support a novel axis of type I IFN-dependent antiviral immunity in the virus-infected brain that is driven by hsp70.

Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.

Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus.

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the µM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.

Role of hepatic PKCß in nutritional regulation of hepatic glycogen synthesis.

The signaling mechanisms by which dietary fat and cholesterol signals regulate central pathways of glucose homeostasis are not completely understood. By using a hepatocyte-specific PKCß-deficient (PKCßHep-/-) mouse model, we demonstrated the role of hepatic PKCß in slowing disposal of glucose overload by suppressing glycogenesis and increasing hepatic glucose output. PKCßHep-/- mice exhibited lower plasma glucose under the fed condition, modestly improved systemic glucose tolerance and mildly suppressed gluconeogenesis, increased hepatic glycogen accumulation and synthesis due to elevated glucokinase expression and activated glycogen synthase (GS), and suppressed glucose-6-phosphatase expression compared with controls. These events were independent of hepatic AKT/GSK-3α/ß signaling and were accompanied by increased HNF-4α transactivation, reduced FoxO1 protein abundance, and elevated expression of GS targeting protein phosphatase 1 regulatory subunit 3C in the PKCßHep-/- liver compared with controls. The above data strongly imply that hepatic PKCß deficiency causes hypoglycemia postprandially by promoting glucose phosphorylation via upregulating glucokinase and subsequently redirecting more glucose-6-phosphate to glycogen via activating GS. In summary, hepatic PKCß has a unique and essential ability to induce a coordinated response that negatively affects glycogenesis at multiple levels under physiological postprandial conditions, thereby integrating nutritional fat intake with dysregulation of glucose homeostasis.

Hepatocyte-specific PKCß deficiency protects against high-fat diet-induced nonalcoholic hepatic steatosis.

OBJECTIVE: Nonalcoholic hepatic steatosis, also known as fatty liver, is a uniform response of the liver to hyperlipidic-hypercaloric diet intake. However, the post-ingestive signals and mechanistic processes driving hepatic steatosis are not well understood. Emerging data demonstrate that protein kinase C beta (PKCß), a lipid-sensitive kinase, plays a critical role in energy metabolism and adaptation to environmental and nutritional stimuli. Despite its powerful effect on glucose and lipid metabolism, knowledge of the physiological roles of hepatic PKCß in energy homeostasis is limited. METHODS: The floxed-PKCß and hepatocyte-specific PKCß-deficient mouse models were generated to study the in vivo role of hepatocyte PKCß on diet-induced hepatic steatosis, lipid metabolism, and mitochondrial function. RESULTS: We report that hepatocyte-specific PKCß deficiency protects mice from development of hepatic steatosis induced by high-fat diet, without affecting body weight gain. This protection is associated with attenuation of SREBP-1c transactivation and improved hepatic mitochondrial respiratory chain. Lipidomic analysis identified significant increases in the critical mitochondrial inner membrane lipid, cardiolipin, in PKCß-deficient livers compared to control. Moreover, hepatocyte PKCß deficiency had no significant effect on either hepatic or whole-body insulin sensitivity supporting dissociation between hepatic steatosis and insulin resistance. CONCLUSIONS: The above data indicate that hepatocyte PKCß is a key focus of dietary lipid perception and is essential for efficient storage of dietary lipids in liver largely through coordinating energy utilization and lipogenesis during post-prandial period. These results highlight the importance of hepatic PKCß as a drug target for obesity-associated nonalcoholic hepatic steatosis.

High affinity binding between Hsp70 and the C-terminal domain of the measles virus nucleoprotein requires an Hsp40 co-chaperone.

The major inducible 70 kDa heat shock protein (hsp70) binds the measles virus (MeV) nucleocapsid with high affinity in an ATP-dependent manner, stimulating viral transcription and genome replication, and profoundly influencing virulence in mouse models of brain infection. Binding is mediated by two hydrophobic motifs (Box-2 and Box-3) located within the C-terminal domain (N(TAIL)) of the nucleocapsid protein, with N(TAIL) being an intrinsically disordered domain. The current work showed that high affinity hsp70 binding to N(TAIL) requires an hsp40 co-chaperone that interacts primarily with the hsp70 nucleotide binding domain (NBD) and displays no significant affinity for N(TAIL). Hsp40 directly enhanced hsp70 ATPase activity in an N(TAIL)-dependent manner, and formation of hsp40-hsp70-N(TAIL) intracellular complexes required the presence of N(TAIL) Box-2 and 3. Results are consistent with the functional interplay between hsp70 nucleotide and substrate binding domains (SBD), where ATP hydrolysis is rate limiting to high affinity binding to client proteins and is enhanced by hsp40. As such, hsp40 is an essential variable in understanding the outcome of MeV-hsp70 interactions.

Gene copy-number variation and associated polymorphisms of complement component C4 in human systemic lupus erythematosus (SLE): low copy number is a risk factor for and high copy number is a protective factor against SLE susceptibility in European Americans.

Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with > or =5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas > or =3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the -308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease.