Ceruloplasmin: Macromolecular Assemblies with Iron-Containing Acute Phase Proteins

Copper-containing ferroxidase ceruloplasmin (Cp) forms binary and ternary complexes with cationic proteins lactoferrin (Lf) and myeloperoxidase (Mpo) during inflammation. We present an X-ray crystal structure of a 2Cp-Mpo complex at 4.7 Å resolution. This structure allows one to identify major protein–protein interaction areas and provides an explanation for a competitive inhibition of Mpo by Cp and for the activation of p-phenylenediamine oxidation by Mpo. Small angle X-ray scattering was employed to construct low-resolution models of the Cp-Lf complex and, for the first time, of the ternary 2Cp-2Lf-Mpo complex in solution. The SAXS-based model of Cp-Lf supports the predicted 1∶1 stoichiometry of the complex and demonstrates that both lobes of Lf contact domains 1 and 6 of Cp. The 2Cp-2Lf-Mpo SAXS model reveals the absence of interaction between Mpo and Lf in the ternary complex, so Cp can serve as a mediator of protein interactions in complex architecture. Mpo protects antioxidant properties of Cp by isolating its sensitive loop from proteases. The latter is important for incorporation of Fe³⁺ into Lf, which activates ferroxidase activity of Cp and precludes oxidation of Cp substrates. Our models provide the structural basis for possible regulatory role of these complexes in preventing iron-induced oxidative damage.     

Increase in Sensitivity of HEK293FT Cells to Influenza Infection by CRISPR-Cas9-Mediated Knockout of IRF7 Transcription Factor

Russian Journal of Bioorganic Chemistry - Interferon-regulated factors play a central role in the activation of the innate immune response. The interferon-regulatory factor 7 (IRF7) is one of the...     

Obtaining mice that carry human mitochondrial DNA transmitted to the progeny

To study human diseases associated with mutations in mitochondrial DNA one needs an animal model in which the distribution of abnormal mtDNA and its impact on the phenotype might be followed. We isolated human mitochondria from HepG2 cell culture and microinjected them into murine zygotes, upon which those were transplanted to the pseudopregnant mice. PCR with species-specific primers allowed detecting human mtDNA in the tissues of 7-13-day embryos. No serious alterations in the development of transmitochondrial embryos were noticed. Among various organs/tissues of the 13-day embryos, human mtDNA was detected only in the heart, skeletal muscles, and stomach, which is in line with its uneven distribution among the blastomeres of an early mouse embryo that we described previously. In four recipient females, the microinjected zygotes were allowed to develop to term, the four neonate males of their joint litter were sacrificed, and in three of them human mtDNA was detected in the heart, skeletal muscles, stomach, brain, testes, and bladder. Six females of that joint litter were grown and mated to intact males. In the progeny (F1) of one of the females two mice were carrying human mtDNA in the heart, skeletal muscles, stomach, brain, lungs, uterus, ovaries, and kidneys. The study confirms the possibility to obtain transmitochondrial mice carrying human mtDNA that is transmitted to the animals of the next generation. Our results also indicate that among the organs to which human mtDNA is distributed some are more likely to receive it than others.     

Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.     

Interaction of ceruloplasmin and 5-lipoxygenase

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300–400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5–10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2–10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed.     

Study of Interaction of Ceruloplasmin with Serprocidins

This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1: 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K _i ≈ 1 μM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K _i ≈20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5–13, 61–63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.     

Zooplankton of the Patagonian slope and adjacent waters in the autumn

In autumn, from March until May 1988 in the Patagonian shelf and slope zones, the system of the water current was modified and water temperature decreased. The biomass of seston and abundance of zooplankton decreased simultaneously, especially those of the subantactric species Calanus simillimus. Zooplankton organisms started their descent to deeper waters. The Antarctic species Rhincalanus gigas was common in the upper layers in March. In May, R. gigas was rare and occurred in deeper layers. Subtropical zooplankton species dominated in the shelf zone, while subantartic species dominated in the slope zone within the Falkland current. In May, the abundance of shelf species in the slope zone increased due to weakening of the Falkland current and its dislocation to the east. The zooplankton biomass and abundance increased at the zone of water divergence in the east and at the slope frontal zone in the west.     

Novel <Emphasis Type="Italic">BRCA1</Emphasis> gene mutations in breast cancer patients from St. Petersburg

Screening of patients with familial breast cancer from St. Petersburg for BRCA1 gene mutations resulted in identification of three mutations (4146del3, 2761delA, and A622V) and two polymorphisms (P871L and S1436S). Mutations 4146del3 and 2761delA are novel, never previously described elsewhere. Deletion 2761delA produces a reading frame shift, premature protein synthesis termination and can cause predisposition for breast cancer. Deletion 4146del3 does not cause a frame shift, but can result not only in the disappearance of amino acid residue (D1343del) in the BRCA1 protein but also in alteration of folding of the protein, entailing loss of its functional activity. Two variants of nucleotide sequence observed in the number of patients were classified as DNA polymorphisms (P871L and S1436S) rather than mutations as they were not tightly associated with the increased risk of breast cancer.