Structural Basis of Importin-alpha-Mediated Nuclear Transport for Ku70 and Ku80

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

Background: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. Objective: the objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. Methods: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Results: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. Conclusion: the microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations

The effects of copper ions on the synthesis of periplasmic and membrane proteins in Acidithiobaeillus ferrooxidans as analyzed by SDS-PAGE and 2D-PAGE

The effects of 200 mM copper ions on the synthesis of membrane and periplasmic proteins were investigated in iron-grown cells of Acidithiobacillus ferrooxidans (At. ferrooxidans). Total membrane protein profiles of cells grown in the absence of copper ions (unadapted cells) and in the presence of copper ions (copper-adapted cells) were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Crude preparations of outer membrane and periplasmic proteins were analyzed by SDS-PAGE. The synthesis of proteins was diminished or increased in the presence of copper ions. Low molecular weight proteins (< 14 kDa) were significantly repressed by copper. These proteins are probably acidic proteins located in the outer membrane. An over-expression of a periplasmic protein of about 17 kDa was detected in the copper-adapted cells and was assumed to be rusticyanin, a 16.5-kDa periplasmic copper protein present in At. ferrooxidans cells and involved in the electron-transport chain of the iron oxidation pathway. To our knowledge, this is the first report of a possible involvement of the rusticyanin and outer membrane proteins in the mechanism of copper resistance in At. ferrooxidans. (C) 2003 Elsevier B.V. All rights reserved.

Mecanismos de absorção de aminoácidos e oligopeptídios. Controle e implicações na dietoterapia humana

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na + or H + co-transporters) pathways. Active Na +-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H + co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na + dependency whereas the later is active H +-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H +-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

Rest stops during road transport: Impacts on performance and acute-phase protein responses of feeder cattle

Angus x Hereford steers (n = 42) and heifers (n = 21) were ranked by gender and BW on d 0 of the experiment and randomly assigned to 1 of 3 treatments: 1) no transport and full access to feed and water (CON); 2) continuous road transport for 1,290 km (TRANS), or 3) road transport for 1,290 km, with rest stops every 430 km (STOP; total of 2 rest stops). Treatments were applied from d 0 to 1 of the experiment. Cattle from TRANS and STOP treatments were transported in separate commercial livestock trailers, within a single 2.1 x 7.2 m compartment, but through the exact same route. During each rest stop, STOP cattle were unloaded and offered mixed alfalfa-grass hay and water for ad libitum consumption for 2 h. Upon arrival of STOP and TRANS on d 1, cattle were ranked by sex and BW within each treatment and assigned to 21 feedlot pens (7 pens/treatment; 2 steers and 1 heifer/pen). Full BW was recorded before (d -1 and 0) treatment application and at the end of experiment (d 28 and 29). Total DMI was evaluated daily from d 1 to 28. Blood samples were collected on d 0 (before loading of TRANS and STOP cattle), 1 (immediately after unloading of TRANS and STOP cattle), 4, 7, 10, 14, 21, and 28. Body weight shrink from d 0 to d 1 was reduced (P < 0.01) in CON compared to TRANS and STOP, and reduced in STOP compared to TRANS. Mean ADG was greater (P < 0.05) in CON compared to TRANS and STOP, but similar (P = 0.68) between TRANS and STOP. No treatment effects were detected (P >= 0.18) on hay, concentrate, and total DMI. Mean G: F was greater (P = 0.05) in CON compared to STOP, tended to be greater (P = 0.08) in CON compared to TRANS, and similar (P = 0.85) between TRANS and STOP. Plasma cortisol concentrations were greater (P <= 0.04) in TRANS compared to CON and STOP on d 1, and greater (P = 0.04) in TRANS compared to CON on d 4. Serum NEFA concentrations were greater (P < 0.01) in TRANS compared to CON and STOP on d 1, and greater (P <= 0.05) in TRANS compared to CON on d 4 and 7. Mean plasma ceruloplasmin concentrations were similar (P = 0.19) among treatments. Plasma haptoglobin concentrations were greater (P <= 0.04) in TRANS compared to CON and STOP on d 1, and in STOP compared to CON on d 1. In conclusion, inclusion of rest stops during a 1,290-km transport prevented the increase in circulating cortisol and alleviated the NEFA and haptoglobin response elicited by transport, but did not improve feedlot receiving performance of transported cattle.

eIF5A has a function in the cotranslational translocation of proteins into the ER

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transport of amino acids from milk whey by Caco-2 cell monolayer after hydrolytic action of gastrointestinal enzymes

The bioavailability of amino adds from milk whey protein hydrolysates was evaluated using diffusion of the substances through semi-permeable membranes (dialyzability) and transport by Caco-2 cell cultures. The hydrolysates with low degree of hydrolysis (LDH) and high degree of hydrolysis (HDH) were obtained after 120 min of reaction time at 50 degrees C after the initial addition of pepsin, followed by the addition of trypsin, chymotrypsin and carboxypeptidase-A. The proteins and hydrolysates were further subjected to in vitro digestion with pepsin plus pancreatin. HPLC was used to determine the concentrations of dialyzable amino adds (48.4% of the non-hydrolyzed proteins, 63.2% of the LDH sample and 58.3% of the HDH sample), demonstrating the greater dialyzability of the hydrolysates. The LDH and HDH whey protein hydrolysates prepared with pepsin, trypsin, chymotrypsin and carboxypeptidase-A showed only 14.7% and 20.8% of dialyzable small peptides and amino acids, respectively. The efficiency of absorption was demonstrated by the preferential transport of Ile, Lou and Arg through a layer of cells. In the LDH hydrolysate, Tyr was also transported. Prior high- and low-degree hydrolysis of the whey provided transport by 5.7% and 6.6%, respectively, in comparison with 23% for non-hydrolyzed proteins, considering the total amount of these amino adds that was applied to the cells. (C) 2014 Elsevier Ltd. All rights reserved.

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.

Uncoupling proteins (UCP) in unicellular eukaryotes: True UCPs or UCP1-like acting proteins?

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K-m 55.7 mu M) in an optimum pH of 7.1, and this activity is effectively retained until 50 degrees C. CeSP remained stable in the presence of kosmotropic anions (PO43-, SO42-, and CH3COO-) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

Abstract Background Heavy metal Resistance-Nodulation-Division (HME-RND) efflux systems help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations. These proteins constitute the cytoplasmic membrane channel of the tripartite RND transport systems. Caulobacter crescentus NA1000 possess two HME-RND proteins, and the aim of this work was to determine their involvement in the response to cadmium, zinc, cobalt and nickel, and to analyze the phylogenetic distribution and characteristic signatures of orthologs of these two proteins. Results Expression assays of the czrCBA operon showed significant induction in the presence of cadmium and zinc, and moderate induction by cobalt and nickel. The nczCBA operon is highly induced in the presence of nickel and cobalt, moderately induced by zinc and not induced by cadmium. Analysis of the resistance phenotype of mutant strains showed that the ΔczrA strain is highly sensitive to cadmium, zinc and cobalt, but resistant to nickel. The ΔnczA strain and the double mutant strain showed reduced growth in the presence of all metals tested. Phylogenetic analysis of the C. crescentus HME-RND proteins showed that CzrA-like proteins, in contrast to those similar to NczA, are almost exclusively found in the Alphaproteobacteria group, and the characteristic protein signatures of each group were highlighted. Conclusions The czrCBA efflux system is involved mainly in response to cadmium and zinc with a secondary role in response to cobalt. The nczCBA efflux system is involved mainly in response to nickel and cobalt, with a secondary role in response to cadmium and zinc. CzrA belongs to the HME2 subfamily, which is almost exclusively found in the Alphaproteobacteria group, as shown by phylogenetic analysis. NczA belongs to the HME1 subfamily which is more widespread among diverse Proteobacteria groups. Each of these subfamilies present distinctive amino acid signatures.

Theoretical studies of Membrane Proteins : Properties, Prediction Methods and Genome-wide analysis

Membrane proteins are a large and important class of proteins. They are responsible for several of the key functions in a living cell, e.g. transport of nutrients and ions, cell-cell signaling, and cell-cell adhesion. Despite their importance it has not been possible to study their structure and organization in much detail because of the difficulty to obtain 3D structures. In this thesis theoretical studies of membrane protein sequences and structures have been carried out by analyzing existing experimental data. The data comes from several sources including sequence databases, genome sequencing projects, and 3D structures. Prediction of the membrane spanning regions by hydrophobicity analysis is a key technique used in several of the studies. A novel method for this is also presented and compared to other methods. The primary questions addressed in the thesis are: What properties are common to all membrane proteins? What is the overall architecture of a membrane protein? What properties govern the integration into the membrane? How many membrane proteins are there and how are they distributed in different organisms? Several of the findings have now been backed up by experiments. An analysis of the large family of G-protein coupled receptors pinpoints differences in length and amino acid composition of loops between proteins with and without a signal peptide and also differences between extra- and intracellular loops. Known 3D structures of membrane proteins have been studied in terms of hydrophobicity, distribution of secondary structure and amino acid types, position specific residue variability, and differences between loops and membrane spanning regions. An analysis of several fully and partially sequenced genomes from eukaryotes, prokaryotes, and archaea has been carried out. Several differences in the membrane protein content between organisms were found, the most important being the total number of membrane proteins and the distribution of membrane proteins with a given number of transmembrane segments. Of the properties that were found to be similar in all organisms, the most obvious is the bias in the distribution of positive charges between the extra- and intracellular loops. Finally, an analysis of homologues to membrane proteins with known topology uncovered two related, multi-spanning proteins with opposite predicted orientations. The predicted topologies were verified experimentally, providing a first example of "divergent topology evolution".

Strong iron demand during hypoxia-induced erythropoiesis is associated with down-regulation of iron-related proteins and myoglobin in human skeletal muscle

[EN] Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

Numerical simulation of ion transport through ion channels and solid-state nanopores

Ion channels are pore-forming proteins that regulate the flow of ions across biological cell membranes. Ion channels are fundamental in generating and regulating the electrical activity of cells in the nervous system and the contraction of muscolar cells. Solid-state nanopores are nanometer-scale pores located in electrically insulating membranes. They can be adopted as detectors of specific molecules in electrolytic solutions. Permeation of ions from one electrolytic solution to another, through a protein channel or a synthetic pore is a process of considerable importance and realistic analysis of the main dependencies of ion current on the geometrical and compositional characteristics of these structures are highly required. The project described by this thesis is an effort to improve the understanding of ion channels by devising methods for computer simulation that can predict channel conductance from channel structure. This project describes theory, algorithms and implementation techniques used to develop a novel 3-D numerical simulator of ion channels and synthetic nanopores based on the Brownian Dynamics technique. This numerical simulator could represent a valid tool for the study of protein ion channel and synthetic nanopores, allowing to investigate at the atomic-level the complex electrostatic interactions that determine channel conductance and ion selectivity. Moreover it will provide insights on how parameters like temperature, applied voltage, and pore shape could influence ion translocation dynamics. Furthermore it will help making predictions of conductance of given channel structures and it will add information like electrostatic potential or ionic concentrations throughout the simulation domain helping the understanding of ion flow through membrane pores.

Analyse der Transportfunktion und Proteinexpression des kationischen Aminosäure-Transporters hCAT-3 und des verwandten Proteins SLC7A4

Die vorliegende Arbeit befasst sich mit der Charakterisierung des humanen kationischen Aminosäure-Transporters 3 (hCAT-3) und mit der Generierung spezifischer Antikörper gegen hCAT-3, mCAT-3 und das verwandte Protein SLC7A4. Im ersten Teil dieser Arbeit wurde gezeigt, dass hCAT-3 glykosyliert und in der Plasmamembran lokalisiert ist. Transportstudien an hCAT-3-exprimierenden Xenopus laevis Oozyten demonstrierten einen selektiven Transport von kationischen L-Aminosäuren. Die Transporteigenschaften von hCAT-3 (KM, Vmax, Trans-Stimulation) ähnelten den der Isoform hCAT-2B am meisten. Diese Ergebnisse stehen im Gegensatz zu Untersuchungen an CAT-3 von Maus und Ratte, in denen nur eine geringe CAT-Aktivität gezeigt wurde, die zudem durch neutrale und anionische Aminosäuren und durch D-Arginin hemmbar war. Die höchste Expression von hCAT-3 wurde im Thymus gefunden, was bedeutet, dass er nicht auf neuronale Zellen beschränkt ist. Dieser Befund steht im Gegensatz zur ausschließlich neuronalen Expression von rCAT-3 bzw. mCAT-3. Ein Schwerpunkt der Arbeit lag in der Generierung spezifischer Antikörper gegen die C-Termini des humanen und murinen CAT-3 und des verwandten Proteins SLC7A4. Dafür wurden Antiseren gegen Fusionsproteine zwischen dem bakteriellen trpE-Protein und den C-Termini der jeweiligen Isoform gewonnen. Zur Aufreinigung der Antikörpern wurden Affinitätssäulen mit Fusionsprotein aus Glutathion S-Transferase und den jeweils gleichen carboxyterminalen Aminosäuren von hCAT-3, SLC7A4 und mCAT-3 hergestellt. Zur Überprüfung der Spezifität der Affinitäts-aufgereinigten Antikörper wurden Western-Blot-Analysen mit Lysaten von X. laevis-Oozyten durchgeführt, die mit cRNA der jeweiligen Isoform injiziert worden waren. Weiterhin wurden humane U373MG Glioblastom-Zellen, in denen die jeweilige Isoform überexprimiert worden war, zur Überprüfung der Antikörper verwendet. Es konnte nachgewiesen werden, dass die neu gewonnenen Antikörper das jeweilige glykosylierte und deglykosylierte CAT-Protein spezifisch erkannten. Die hCAT-3 Antikörper wurden dazu verwendet die endogene Expression in NT2-Teratokarzinom-Zellen nachzuweisen. Hierbei zeigte sich, dass die intrazelluläre Expression höher war als an der Zelloberfläche. Nur etwa 10-20% des hCAT-3-Gesamtproteins wurden an der Zelloberfläche exprimiert. Die gleiche subzelluläre Verteilung zeigte sich in mit hCAT-3.EGFP stabil transfizierten U373MG-Zellen. Um die Auswirkung einer PKC-Aktivierung auf die Aktivität und Expression von hCAT-3 untersuchen zu können, wurden Transportexperimente an Oozyten und Western-Blot Analysen mit biotinylierten Zelloberflächen-Proteinen durchgeführt. Der PKC-Aktivator PMA reduzierte die hCAT-3 Expression um ca. 35% reduziert. Sowohl in X. laevis Oozyten, als auch in U373MG-Zellen war die Verminderung der Transportaktivität von einer Reduktion der Zelloberflächen-Expression von hCAT-3 begleitet. Die Vorbehandlung mit dem PKC-Inhibitor Bisindolylmaleimid I (BIM I) reduzierte beide PMA-Effekt. Es ist daher davon auszugehen, dass die Reduktion der Zelloberflächen-Expression durch PKC vermittelt wurde. Ähnlich wie hCAT-1 scheint auch hCAT-3 durch eine klassische PKC, am wahrscheinlichsten PKC? und PKC?, herunterreguliert zu werden. Durch konfokale Mikroskopie von überexprimierten hCAT-3.GFP-Konstrukten in U373MG-Zellen konnten diese Ergebnisse bestätigt werden. Ähnliche Ergebnisse wurden auch unter Verwendung der selbst hergestellten Antikörper gegen den endogenen hCAT-3 in NT2 Teratokarzinom-Zellen erzielt.