Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

Binding properties of YjeQ (RsgA), RbfA, RimM and Era to assembly intermediates of the 30S subunit.

Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells. We measured by microscale thermophoresis that YjeQ and Era binds to the mature 30S subunit with high affinity. However, the binding affinity of these factors to the immature particles and of RimM and RbfA to mature or immature particles was weak, suggesting that binding is not occurring at physiological concentrations. These results suggest that in the absence of these factors, the immature particles evolve into a thermodynamically stable intermediate that exhibits low affinity for the assembly factors. These results imply that the true substrates of YjeQ, RbfA, RimM and Era are immature particles that precede the ribosomal particles accumulating in the knockouts strains.

Yeast rvb1 and rvb2 proteins oligomerize as a conformationally variable dodecamer with low frequency.

Rvb1 and Rvb2 are conserved AAA+ (ATPases associated with diverse cellular activities) proteins found at the core of large multicomponent complexes that play key roles in chromatin remodeling, integrity of the telomeres, ribonucleoprotein complex biogenesis and other essential cellular processes. These proteins contain an AAA+ domain for ATP binding and hydrolysis and an insertion domain proposed to bind DNA/RNA. Yeast Rvb1 and Rvb2 proteins oligomerize primarily as heterohexameric rings. The six AAA+ core domains form the body of the ring and the insertion domains protrude from one face of the ring. Conversely, human Rvbs form a mixture of hexamers and dodecamers made of two stacked hexamers interacting through the insertion domains. Human dodecamers adopt either a contracted or a stretched conformation. Here, we found that yeast Rvb1/Rvb2 complexes when assembled in vivo mainly form hexamers but they also assemble as dodecamers with a frequency lower than 10%. Yeast dodecamers adopt not only the stretched and contracted structures that have been described for human Rvb1/Rvb2 dodecamers but also intermediate conformations in between these two extreme states. The orientation of the insertion domains of Rvb1 and Rvb2 proteins in these conformers changes as the dodecamer transitions from the stretched structure to a more contracted structure. Finally, we observed that for the yeast proteins, oligomerization as a dodecamer inhibits the ATPase activity of the Rvb1/Rvb2 complex. These results indicate that although human and yeast Rvb1 and Rvb2 proteins share high degree of homology, there are significant differences in their oligomeric behavior and dynamics.

Development, antibiotic production, and ribosome assembly in Streptomyces venezuelae are impacted by RNase J and RNase III deletion.

RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics.

Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement.

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an "early convergency model" in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.

Factors defining the functional oligomeric state of Escherichia coli DegP protease.

Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP.

Registration for public drug benefits across areas of differing ethnic composition in British Columbia, Canada.

BACKGROUND: In 2003, the government of British Columbia, Canada introduced a universal drug benefit plan to cover drug costs that are high relative to household income. Residents were required to register in order to be eligible for the income-based benefits. Given past research suggesting that registration processes may pose an access barrier to certain subpopulations, we aimed to determine whether registration rates varied across small geographic areas that differed in ethnic composition. METHODS: Using linked population-based administrative databases and census data, we conducted multivariate logistic regression analyses to determine whether the probability of registration for the public drug plan varied across areas of differing ethnic composition, controlling for household-level predisposing, enabling and needs factors. RESULTS: The adjusted odds of registration did not differ across regions characterized by high concentrations (greater than 30%) of residents identifying as North American, British, French or other European. Households located in areas with concentrations of residents identifying as an Asian ethnicity had the highest odds of program registration: Chinese (OR = 1.21, CI: 1.19-1.23) and South Asian (OR = 1.19, CI: 1.16-1.22). Despite this positive finding, households residing in areas with relatively high concentrations of recent immigrants had slightly lower adjusted odds of registering for the program (OR = 0.97, CI: 0.95-0.98). CONCLUSIONS: This study identified ethnic variation in registration for a new public drug benefit program in British Columbia. However, unlike previous studies, the variation observed did not indicate that areas with high concentrations of certain ethnicities experienced disadvantages. Potential explanations are discussed.

Nuclear import of human MLH1, PMS2, and MutLalpha: redundancy is the key.

DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.

Costs and determinants of privately financed home-based health care in Ontario, Canada.

The Canadian context in which home-based healthcare services are delivered is characterised by limited resources and escalating healthcare costs. As a result, a financing shift has occurred, whereby care recipients receive a mixture of publicly and privately financed home-based services. Although ensuring that care recipients receive efficient and equitable care is crucial, a limited understanding of the economic outcomes and determinants of privately financed services exists. The purposes of this study were (i) to determine costs incurred by families and the healthcare system; (ii) to assess the determinants of privately financed home-based care; and (iii) to identify whether public and private expenditures are complements or substitutes. Two hundred and fifty-eight short-term clients (<90 days of service utilisation) and 256 continuing care clients (>90 days of utilisation) were recruited from six regions across the province of Ontario, Canada, from November 2003 to August 2004. Participants were interviewed by telephone once a week for 4 weeks and asked to provide information about time and monetary costs of care, activities of daily living (ADL), and chronic conditions. The mean total cost of care for a 4-week period was $7670.67 (in 2004 Canadian dollars), with the overwhelming majority of these costs (75%) associated with private expenditures. Higher age, ADL impairment, being female, and a having four or more chronic conditions predicted higher private expenditures. While private and public expenditures were complementary, private expenditures were somewhat inelastic to changes in public expenditures. A 10% increase in public expenditures was associated with a 6% increase in private expenditures. A greater appreciation of the financing of home-based care is necessary for practitioners, health managers and policy decision-makers to ensure that critical issues such as inequalities in access to care and financial burden on care recipients and families are addressed.

Role of the PDZ domains in Escherichia coli DegP protein.

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.

The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity.

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28 degrees C, but efficiently degrading unfolded proteins above 28 degrees C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.

The Magnitude, Share and Determinants of Private Costs Incurred by Clients (and Their Caregivers) of In-home Publicly Financed Care.

Home-based health services remain one of the fastest-growing sectors in the Canadian healthcare system. While there have been studies addressing the characteristics of home care users and the determinants of utilization, the costs associated with the use of home care services, particularly private costs, have been largely neglected. To gain a comprehensive appreciation of the financing context in which ambulatory and home-based care is delivered and received, it is imperative to assess costs incurred by clients and their family/friends. Accordingly, this study examined the magnitude and determinants of the share of private costs incurred by Ontarians who received in-home publicly financed services and by their unpaid caregivers. The private share of costs was found to be 75%. Determinants of the private share included participants' gender, martial status, functioning in activities of daily living and the type and length of service received. These findings suggest that recipients of home-based health services in Ontario may bear an economic burden when care is shifted into the home setting.

Mapping of functional regions in the amino-terminal portion of the herpes simplex virus ICP27 regulatory protein: importance of the leucine-rich nuclear export signal and RGG Box RNA-binding domain.

Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.