Ferritoid,a tissue-specific nuclear transport protein for ferritin in corneal epithelial cells

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of approximately 50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.     

Role of protons in the thermodynamic contribution of a Zn(II)-Cys4 site toward metalloprotein stability

The current limited understanding of the free energy contributions of metal-protein interactions toward metalloprotein stability is largely due to an inability to separate the energetics of the metal-ligand and protein-protein interactions. In order to elucidate the thermodynamic contribution of a Zn(II)-(S.Cys)4 site toward metalloprotein stability relevant to classic structural Zn(II) sites, the reaction of {Zn(II)(H2O)6}2+ with a minimal, unstructured, tetracysteine 16-mer peptide, GGG, is described. Isothermal titration fluorimetry over the pH range of 4.5 to 9.0 is used to measure the free energy of Zn(II) binding to the model peptide GGG. The data show that, in the absence of proton competition, Zn(II) binds to the Cys4 coordination sphere with a Kd of 60 aM, indicating that the Zn(II)-(S.Cys)4 interaction can provide up to 22.1 kcal mol-1 in driving force for protein stabilization, folding, and/or assembly. Isothermal titration calorimetry shows that Zn(II)-GGG formation is entropy driven because of water release from both the metal and the peptide scaffold. At pH 7.0, where the Zn(II)-GGG Kd value is 8.0 pM, the reaction releases 3.8 protons, is endothermic with DeltaHrxn of +6.4 kcal mol-1, and entropy driven with DeltaSrxn of +72 cal K-1 mol-1. At pH 8.0, where the peptide is partially deprotonated prior to Zn(II) binding, the 1.0 fM Zn(II)-GGG Kd value reflects a Zn(II) complexation reaction involving the release of 2.5 protons, which is slightly exothermic, with DeltaHrxn of -2.0 kcal mol-1, and largely entropy driven, with DeltaSrxn of +61 cal K-1 mol-1. At pH 5.5, where proton competition weakens the Kd to 4.0 microM, only 3.2 protons are released upon Zn(II) binding, the reaction is endothermic, with DeltaHrxn of +7.7 kcal mol-1, and entropy driven, with DeltaSrxn of +51 cal K-1 mol-1. Likely an intrinsic property of Zn(II)-(S.Cys)4 sites, the entropy driven binding of Zn(II) reflects the proton dependent chemical speciation of the Zn(II)-(S.Cys)4 peptide complex and its effects on modulating the dehydration of both the peptide and metal. Furthermore, the Zn(II) binding thermodynamics of a variety of Zn(II) proteins at pH 7.0 reveals the presence of enthalpy-entropy compensation (EEC) phenomena in nature.     

Growth hormone and testosterone interact positively to enhance protein and energy metabolism in hypopituitary men

We investigated the impact of growth hormone (GH) alone, testosterone (T) alone, and combined GH and T on whole body protein metabolism. Twelve hypopituitary men participated in two studies. Study 1 compared the effects of GH alone with GH plus T, and study 2 compared the effects of T alone with GH plus T. IGF-I, resting energy expenditure (REE), and fat oxidation (F(ox)) and rates of whole body leucine appearance (R(a)), oxidation (L(ox)), and nonoxidative leucine disposal (NOLD) were measured. In study 1, GH treatment increased mean plasma IGF-I (P < 0.001). GH did not change leucine R(a) but reduced L(ox) (P < 0.02) and increased NOLD (P < 0.02). Addition of T resulted in an additional increase in IGF-I (P < 0.05), reduction in Lox (P < 0.002), and increase in NOLD (P < 0.002). In study 2, T alone did not alter IGF-I levels. T alone did not change leucine R(a) but reduced L(ox) (P < 0.01) and increased NOLD (P < 0.01). Addition of GH further reduced L(ox) (P < 0.05) and increased NOLD (P < 0.05). In both studies, combined treatments on REE and F(ox) were greater than either alone. In summary, GH-induced increase of circulating IGF-I is augmented by T, which does not increase IGF-I in the absence of GH. T and GH exerted independent and additive effects on protein metabolism, F(ox) and REE. The anabolic effects of T are independent of circulating IGF-I.     

Cobalt(II) and zinc(II) binding to a ferredoxin maquette

We have examined the Co(II) and Zn(II) affinity of the prototype ferredoxin maquette ligand, NH(2)-KLCEGG.CIACGAC.GGW-CONH2 (IAA), which was originally designed to bind a [4Fe-4S] cluster. UV-Vis spectroscopy demonstrates tight 1:1 complex formation between Co(II) and IAA. The intensity of the S-->Co(II) charge transfer bands at 304 and 340 nm and the ligand field bands between 630 and 728 nm indicate Co(II) coordination by the four cysteine thiolates of IAA in a pseudo-tetrahedral geometry. A dissociation constant value of 5.3 microM was determined for the Co(II)-IAA complex at pH 6.5. Zn(II) readily displaces Co(II) from IAA as evinced by loss of the Co(II) spectral features. The dissociation constant for Zn(II), 20 pM at pH 6.5, was determined be competition experiments with Co(II)-IAA. These results demonstrate that the ferredoxin maquette ligand is an excellent ligand for Zn(II).     

Study of the Individual Cytochrome b5 and Cytochrome b5 Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

NADH cytochrome b₅ oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b₅ (b₅), CHORD-SGT1 (CS), and cytochrome b₅ reductase (b₅R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b₅ and b₅R domains (Ncb5or-b₅ and Ncb5or-b₅R, respectively) and compared them with human microsomal b₅ (Cyb5A) and b₅R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b₅ reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His⁸⁹ and His¹¹², consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b₅ family shown to have such a heme environment. Like other b₅ family members, Ncb5or-b₅ has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b₅ differs from Cyb5A with respect to location of the second heme ligand (His¹¹²) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b₅R to Ncb5or-b₅ is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b₅ and b₅R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b₅R domains suggest that the CS domain facilitates docking of the b₅ and b₅R domains. Trp¹¹⁴ is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b₅R domain to the b₅ domain.     

Wear Testing of Moderate Activities of Daily Living Using In Vivo Measured Knee Joint Loading

Resumption of daily living activities is a basic expectation for patients provided with total knee replacements. However, there is a lack of knowledge regarding the impact of different activities on the wear performance. In this study the wear performance under application of different daily activities has been analyzed. In vivo load data for walking, walking downstairs/upstairs, sitting down/standing up, and cycling (50 W & 120 W) has been standardized for wear testing. Wear testing of each activity was carried out on a knee wear simulator. Additionally, ISO walking was tested for reasons of comparison. Wear was assessed gravimetrically and wear particles were analyzed. In vivo walking produced the highest overall wear rates, which were determined to be three times higher than ISO walking. Moderate wear rates were determined for walking upstairs and downstairs. Low wear rates were determined for standing up/sitting down and cycling at power levels of 50 W and 120 W. The largest wear particles were observed for cycling. Walking based on in vivo data has been shown to be the most wear-relevant activity. Highly demanding activities (stair climbing) produced considerably less wear. Taking into account the expected number of loads, low-impact activities like cycling may have a greater impact on articular wear than highly demanding activities.