Structural distinction between soluble and particulate protein kinase C species

A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca²⁺/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more -helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200–250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.     

The Price of a Neglected Zoonosis: Case-Control Study to Estimate Healthcare Utilization Costs of Human Brucellosis

Human brucellosis has reemerged as a serious public health threat to the Bedouin population of southern Israel in recent years. Little is known about its economic implications derived from elevated healthcare utilization (HCU). Our objective was to estimate the HCU costs associated with human brucellosis from the insurer perspective. A case-control retrospective study was conducted among Clalit Health Services (CHS) enrollees. Brucellosis cases were defined as individuals that were diagnosed with brucellosis at the Clinical Microbiology Laboratory of Soroka University Medical Center in the 2010–2012 period (n = 470). Control subjects were randomly selected and matched 1:3 by age, sex, clinic, and primary physician (n = 1,410). HCU data, demographic characteristics and comorbidities were obtained from CHS computerized database. Mean±SD age of the brucellosis cases was 26.6±17.6 years. 63% were male and 85% were Bedouins. No significant difference in Charlson comorbidity index was found between brucellosis cases and controls (0.41 vs. 0.45, respectively, P = 0.391). Before diagnosis (baseline), the average total annual HCU cost of brucellosis cases was slightly yet significantly higher than that of the control group ($439 vs. $382, P<0.05), however, no significant differences were found at baseline in the predominant components of HCU, i.e. hospitalizations, diagnostic procedures, and medications. At the year following diagnosis, the average total annual HCU costs of brucellosis cases was significantly higher than that of controls ($1,327 vs. $380, respectively, P<0.001). Most of the difference stems from 7.9 times higher hospitalization costs (p<0.001). Additional elevated costs were 3.6 times higher laboratory tests (P<0.001), 2.8 times higher emergency room visits (P<0.001), 1.8 times higher medication (P<0.001) and 1.3 times higher diagnostic procedures (P<0.001). We conclude that human brucellosis is associated with elevated HCU costs. Considering these results in cost-effective analyses may be crucial for both reducing health inequities and optimal allocation of health systems’ scarce resources.     

Intrinsically active variants of all human p38 isoforms

The p38 mitogen-activated protein kinases are activated in response to various extracellular signals in eukaryotic cells and play a critical role in the cellular responses to these signals. The four mammalian isoforms (p38alpha, p38beta, p38gamma, and p38delta) are coexpressed and coactivated in the same cells. The exact role of each p38 isoform has not been entirely identified, in part due to the inability to activate each member individually. This could be resolved by the use of intrinsically active mutants. Based on previous studies on yeast p38/Hog1 [Bell M, Capone R, Pashtan I, Levitzki A & Engelberg D (2001) J Biol Chem276, 25351-2538] and human p38alpha[Diskin R, Askari N, Capone R, Engelberg D & Livnah O (2004) J Biol Chem279, 47040-47049] we have generated intrinsically active p38beta, p38gamma and p38delta mutants. In addition, we have identified a new activating mutation site in p38alpha. Most of the activating mutations are located in the L16 loop, in which conformational changes were shown to induce activation. We show that these changes impose substantial autophosphorylation activity, providing a mechanistic explanation for the intrinsic activity of the mutants. The new active variants maintain specificity towards substrates and inhibitors similar to that of the parental wild-type proteins, and are phosphorylated by mitogen-activated protein kinase kinase 6, their upstream activator. Thus, we have completed the development of a series of intrinsically active mutants of all p38 isoforms. These active variants could now become powerful tools for the elucidating the activation mechanism and specific biological roles of each p38 isoform.     

Expression, purification, and characterization of recombinant NOD1 (NLRC1): A NLR family member

NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (γ-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery.     

Nbr1 is a novel inhibitor of ligand-mediated receptor tyrosine kinase degradation

Neighbor of BRCA1 (Nbr1) is a highly conserved multidomain scaffold protein with proposed roles in endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic α-helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function.     

Biomechanical stimulation effects on the metabolism of adipocyte

Adipose tissue plays a leading role in obesity, which, in turn, can lead to Type 2 diabetes. Adipocytes (AD) respond to the biomechanical stimulation experienced in fat tissue under static stretch during prolonged sitting or lying. To investigate the effect of such chronic stimulation on adipocyte cell metabolism, we used an in vitro system to mimic the static stretch conditions. Under in vitro culture stretching, cells were analyzed at the single-cell level and we measured an increase in the projected area of the AD and higher content of lipid droplets. A decrease in the projected area of these cells’ nucleus is associated with peroxisome proliferator-activated receptor-gamma expression and heterochromatin. This is the first study to reveal proteins that were altered under static stretch following a mass spectrometry analysis and main pathways that affect cell fate and metabolism. Bioinformatics analysis of the proteins indicated an increase in mitochondrial activity and associated pathways under static stretch stimulation. Quantification of the mitochondrial activity by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the ATPase related proteins specifically measured ATP5B indicated an increase in adipogenesis which points to a higher rate of cell metabolism under static stretch. In summary, our results elaborate on the metabolism of AD exposed to biomechanical stimulation, that is, associated with altered cellular protein profile and thereby influenced cell fate. The static stretch stimulation accelerated adipocyte differentiation through increased mitochondrial activity. Hence, in this study, we introduce a new perspective in understanding the molecular regulation of mechano-transduction in adipogenesis.