Mitochondrion-dependent N-terminal Processing of Outer Membrane Mcl-1 Protein Removes an Essential Mule/Lasu1 Protein-binding Site * □ S
Received for publication, January 5, 2011, and in revised form, May 20, 2011 Published, JBC Papers in Press, May 25, 2011, DOI 10.1074/jbc.M111.218321 Matthew R. Warr ‡1,2,3 , John R. Mills ‡1,4 , Mai Nguyen ‡1 , Stephanie Lemaire-Ewing ‡5 , Jason Baardsnes § , Karen L. W. Sun ‡6 , Abba Malina ‡7 , Jason C. Young ‡ , Danny V. Jeyaraju ¶ , Maureen O’Connor-McCourt § , Luca Pellegrini ¶ , Jerry Pelletier ‡储2 , and Gordon C. Shore ‡储8
For large scale proteinbinding affinity studies, prediction models may be classified into two classes (see also the supplemental Section Methods used in Previous Studies). The first class consists of models using a small number of variables aiming at explaining intuitively important components of the affinity. Based on the observed correlation between the buried surface area (BSA) at the interface and binding affinity , a model splitting the BSA into polar and apolar components was first proposed . A refinement of BSA models with a term coding the depth of interface atoms, called the Voronoi shelling order, was proposed , yielding improvements in particular for rigid cases. The previous models focusing on interfacial properties only, terms coding the percentage of charged and polar a.a. on the interacting surface (NIS) were introduced in , and their connexion with solvent dynamics investigated in . Finally, a model also taking into account the iRMSD, namely the root-mean-square displacement of the Cα atoms of interfacial residues between the bound and unbound states, was recently proposed .
The recent application of convolutional neural networks ( LeCun et al., 2015 ; Salakhutdinov, 2015 ) to sequence-based problems in genomics signals the advent of the deep learning era in computa- tional biology. Two recent methods, DeepBind ( Alipanahi et al., 2015 ) and DeepSEA ( Zhou and Troyanskaya, 2015 ), successfully applied deep learning to modeling the sequence specificity of proteinbinding with a performance superior to the best existing conventional learning methods. Learning tasks in genomics often have tens of thousands or more training examples, which makes them well adapted to training convolutional neural networks without overfitting. Such training examples are typically drawn from high-throughput data, such as those produced by the Encyclopedia of DNA Elements (ENCODE) project ( Bernstein et al., 2012 ).
DnaJ (Hsp40 Protein) Binding to Folded Substrate Impacts
KplE1 Prophage Excision Efficiency * □ S
Received for publication, December 7, 2011, and in revised form, February 23, 2012 Published, JBC Papers in Press, February 28, 2012, DOI 10.1074/jbc.M111.331462
Tania M. Puvirajesinghe ‡1 , Latifa Elantak § , Sabrina Lignon ¶ , Nathalie Franche ‡ , Marianne Ilbert 储 ,
In the frame of this PhD work, we have developed a cost-effective nanofluidic biosensor platform for real-time monitoring of proteinbinding kinetics in physiological media. Our approach exploited biofunctionalized planar extended-nanochannels or nanoslits in combination with a conventional fluorescence microscope, enabling the quantification of kinetic parameters of protein- protein interactions. The nanoslits exhibit several unique characteristics which cannot be attained in typical microfluidic formats. A considerable reduction of the fluorescence background signal within the nanoconfined-analytical space permits real-time monitoring of protein association and dissociation processes without the need of reagent wash and without using sophisticated techniques, such as SPR or TIRF. Furthermore thanks to the molecular confinement between the analytes in solution and the immobilized-probes on the surface, the binding reactions in the nanoslits can be dramatically enhanced, leading to a maximized target capture efficiency and optimized response times. In this framework, we have fabricated nanofluidic devices and conducted kinetic studies of protein interactions. In order to validate our platform, our sensor performances were compared with the existing technology, such as SPR. The outcomes of this study bridging technological and biological aspects are summarized below.
complex (1) are used as a simple model system for the description of a new approach to the labelling polypeptides with ﬂuorescent tags. The strategy takes advantage of the reaction of an acridine orange-based ﬂuorophore (AO) with the non-native metal fragment 1 hybridized on the enzyme. A synthetic methodology for the quantitative metallation of the protein is ﬁrst described and it is then shown that the exogenous metal complex can be exploited for the coupling of the ﬂuorescent probe. All Lys-derived species were characterized by various spectroscopic techniques. It is shown that the approach does not signiﬁcantly alter the activity of the ﬁnal ﬂuorescent metallo-protein conjugate (Lys2). The accumulation of Lys2 on Micrococcus lysodeikticus bacteria was observed via confocal laser scanning microscopy.
fibroblasts both in vitro in three dimensional (3D) matrices, and in athymic nude mice [53,
105]. These findings suggest that pro-cath-D may act as an extracellular bindingprotein by
directly or indirectly triggering an as-yet unidentified cell surface receptor. Our unpublished
results also indicate that pro-cath-D hypersecreted by cancer cells triggers fibroblast invasive
Binding and Serum Resistance 䌤
Sanjay Ram, 1 * Jutamas Ngampasutadol, 1 Andrew D. Cox, 2 Anna M. Blom, 3 Lisa A. Lewis, 1
Frank St. Michael, 2 Jacek Stupak, 2 Sunita Gulati, 1 and Peter A. Rice 1
Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 1 ;
order to extend the application domain of the AFM to ssDNA and its partner proteins. It is generally known that the addition of divalent cations to the deposition buﬀer overcomes the natural repulsion between the negatively charged DNA and the negatively charged mica surface, which leads to the adsorption of dsDNA (24–26) or dsDNA nucleoprotein ﬁlaments on mica (16,18). However ssDNA–SSB nucleoprotein ﬁlaments cannot be properly adsorbed by using the same method. The adsorption mediated by multivalent counterion correlations at the polyelectrolyte–mica interface (27) decreases sharply with the decrease of the polyelectrolyte surface charge density. Thus a loose binding of ssDNA– SSB complexes to mica, resulting from both the ssDNA neutralization and the enlargement of the diameter of the complex upon SSB binding, may explain the unsatisfactory result.
same fixative, rinsed in veronal buffer containing 20% sucrose and frozen in Tissue-Tek (Miles Scientific, Naperville, IL). Coronal sections of 10 mm were incubated overnight at room temperature with a primary antibody directed against either tPA (rabbit anti-tPA, 1:5000; from Pr. Carmeliet, University of Leuven, Belgium), DBP (rabbit anti-DBP, 1:600; Abcam), Microtubule-associated protein 2 (MAP-2; chicken anti-MAP-2, 1:6000; Abcam) or Glial Fibrillary Acidic Protein (GFAP; rabbit anti-GFAP, 1:1000; Sigma). Detection was performed using the corresponding fluorescein isothiocyanate (FITC, green) or tetramethyl rhodamine isothiocyanate (TRITC, red)—conjugated donkey anti IgG secondary antibody (1:300, Jackson Immunoresearch, West Grove, USA). Before being cover slipped with antifed medium containing DAPI, sections were incubated for 5 min with a solution of Sudan Black B (Sigma-Aldrich) in 20% alcohol to reduce the autofluorescence observed in oldest mice (Schnell et al., 1999). Sections were examined with a Leica DM6000 microscope. Images were digitally captured using a coolsnap camera and visualized with Metavue software. DAPI positive cells were quan- tified by the application software Meta Imaging Series 6.3. Values are the means of 10 serial sections for each animal (n = 3).
Tableau 7 : Amorces utilisées pour la mutation silencieuse de la partie de la séquence codante de SBP1 reconnue par le micro ARN artificiel.
Vecteurs codant pour l’expression des protéines GFP-SBP1 et SBP1-GFP
La technique de clonage utilisée pour générer le vecteur codant pour la protéine GST-SBP1 est appelée « clonage Gateway ». Cette méthode permet une recombinaison entre les séquences nucléotidiques attR1 et attR2 contenues dans un clone entrée et les séquences attL1 et attL2 contenues dans un vecteur « destination ». Ceci permet un clonage directionnel de n’importe quel fragment nucléotidique, placé entre attR1 et attR2, dans n’importe quel vecteur « destination ». L’ADNc codant pour SBP1 (U15803, fourni par le National Arabidopsis Resource Center) présent dans un clone « entrée » a été cloné dans un vecteur « destination » (pK7WGF2 fourni par le Flandres Interuniversity Institute for Biotechnology) en aval du promoteur 35S et du gène codant pour le Green Fluorescente Protein (GFP). Concernant SBP1-GFP, le vecteur codant pour celle-ci a été obtenue par une technique de clonage classique qui utilise des enzymes de restriction. La séquence codante pour SBP1 a été amplifiée par PCR et les amorces utilisées ont permis l’ajout d’un site XhoI en amont du codon start en N terminal et un site SpeI en C terminal. L’amorce utilisée pour l’ajout du site SpeI supprime le codon stop et permet ainsi de réaliser une fusion traductionnelle en N terminal de SBP1. L’insert codant pour SBP1 est sous cloné dans le vecteur pGEM-Teasy, digéré par XhoI et SpeI, puis inséré dans le vecteur pGFP-Harper (cordialement fourni par J. Harper, The Scripps Research Institut). Ce vecteur permet, sous le contrôle du promoteur 35S, l’insertion de SBP1 qui ne possède pas de codon stop en amont du gène codant pour la GFP.
Critical Residues at the C-terminal Domain of ABP1 for Its Action at the Plasma Membrane—In addition to the KDEL
sequence, the C-terminal domain of ABP1 contains three highly conserved residues, namely Asp 174 , Glu 175 , and Cys 177 . By using synthetic peptides corresponding to the last 12 or 15 C-terminal amino acids of Nt-abp1, we have suggested previ- ously (30) that the two acidic residues could play an important role in the activation of the electrical response of tobacco pro- toplasts. To overcome the difficulties inherent in the use of short synthetic peptides instead of full-length proteins, we have introduced single or multiple mutations on these residues to determine their importance in the protein as a whole. Pro- teins carrying such mutations showed distinct effects on the electrical response of tobacco protoplasts. In particular, the electrical response was abolished in the case of ABP1-NQ in which Asp 175 and Glu 176 have been converted into their corre- sponding amine (not shown). The suppression of the two neg- ative charges of the acidic residues was responsible for the functional inactivation of the protein. Conversely, the mutation of only one of these residues still produced a response, although weaker than using the wild-type protein. These results suggest that the negative charges upstream of Cys 177 are important either for the interaction of the protein with the plasma mem- brane or for the folding of the protein.
To test the versatility of the scaffold protein to host exogenous epitopes, we selected four different peptide segments of varying size from surface-exposed regions of the N. meningitidis strain M982 TbpA to display. Using the structure of meningococcal TbpA in complex with human Tf as a guide ( 26 ), we selected three regions from the extracellular loops extending off the beta- barrel and one from the plug region (Figure 1B) as these regions appeared to be surface exposed and potentially important for TbpA functionality. The two largest inserts consisted of the majority of the sequence of external loops 10 (30 residues) and 11 (27 residues), best representing the effective transplant of substantial segments of external loop regions. The TbpA loop 3 alpha-helix was inserted into the TbpB loop 20 site in which the anchoring β-strands are not adjacent so that the alpha-helical region could be accommodated. Insertion of these individual regions (Figure 1C) or a combination of the four loop insertions
KEYWORDS: Penicillin binding proteins, boronic acids, antibiotics, antibiotic-resistance, β-lactams, transpeptidase-inhibition
P enicillin-binding proteins (PBPs) catalyze steps in the bio- synthesis of peptidoglycan, which is a major component of the bacterial cell wall (for reviews, see refs 1 and 2), and their inhibition causes irregularities in cell wall structure, lysis, and eventual cell death. 3 PBPs are inhibited by the β-lactam anti- biotics (including penicillins, cephalosporins, monobactams, and carbapenems, Figure 1a). From the 1940s onward, very sub- stantial synthetic and screening eﬀorts were made to optimize the side chains of β-lactam antibiotics with a view to improving their potency, spectrum of activity, and pharmacokinetics (for review, see ref 4). The majority of this work was carried out in the absence of detailed structural knowledge on PBPs, which has begun to emerge over the past decade or so. 1,2,5
Pab2 was found to crosslink to the entire coding region of several Pol II genes. Interestingly, Pab2 showed greater enrichment at the 3’-end as compared to the 5’-end o f genes (Fig. ID). The simplest explanation for the increase association o f Pab2 during transcription elongation is that Pab2 is recruited by or rapidly transferred to the nascent mRNP. This interpretation is consistent with the sensitivity o f Pab2 ChIP and immunoprécipitation assays to RNase, suggesting that Pab2 is cotranscriptionally recruited via the nascent mRNP. The physical fragmentation o f RNA during the sonication step is likely to be the cause for the plateau in ChIP signal detected for Pab2 beyond the middle o f the ADH1, P Y K l, PGK1, and MET26 genes, and reflects the size of chromatin fragments to <500 bp. Interestingly, our ChIP assays indicated that Pab2 occupancies upstream o f the polyadenylation site are sensitive to RNases (Fig. 4). These results could imply that the recruitment o f Pab2 is mediated by direct binding to non-poly(A) sequences in the pre- mRNA prior to polyadenylation. We do not favor this interpretation, however, as studies using nuclear poly(A)-binding proteins from various organisms indicate poor binding to nonpolyadenylated RNA as compared to poly(A) (27,56,57). Analogous to our ChIP experiments, sensitivity o f ChIP signals to RNases was previously reported for the budding yeast proteins Sub2 and Gbp2 (37,58). In this case, results suggest that the TREX complex mediates the cotranscriptional recruitment o f Sub2 and Gbp2 to nascent mRNPs. Similarly, we predict a model in which Pab2 is recruited to the nascent mRNP via protein interactions. A structure-function analysis o f S. pombe Pab2 should provide useful insights into the mechanism that mediates the cotranscriptional recruitment o f Pab2 to nascent mRNPs.
In addition to its incorporation into selenoproteins, selenium can be bound to proteins belonging to the selenium-bindingprotein family (SBP). 3 Many of the beneficial impacts of sele-
nium on mammalian health have been attributed to its role as a critical constituent of selenoproteins and to its binding to SBP1. Although the function of selenoproteins is well established, the activity of SBP1 proteins is still unclear and the link between selenium binding and SBP1 function has not yet been deter- mined. Mammalian SBP1 was first identified in mouse liver (14) in experiments designed to find new selenoproteins. Two homologues are present in humans. Today, SBP genes have been identified in many organisms including plants (14 –18) and additional sequences of SBP homologues are available in public databases from many additional organisms. In humans, down-regulation of SBP1 expression has been correlated with rapid tumor development in many organs (19 –27) and SBP1
Odorant binding proteins, which are abundant low-mol. wt. soluble proteins secreted by the olfactory epithelium in the nasal mucus of vertebrates, play a carrier role for conveying odorants, which are commonly hydrophobic molecules, to their neuronal receptors through nasal mucus (24). In present report a novel rat odorant-bindingprotein variant OBP-1F was used for the preparation of mixed protein/amphiphile LB films. The experiments were performed in the subphase of phosphate buffer solution (PBS) at a pH of 7.5. Under the condition, OBP-1F is perfectly soluble and active. Fatty amine octadecylamine (ODA) was chose as amphiphile taking into account of the fact that at such a pH OBP-1F could be considerably better adsorbed onto positively charged lipid monolayers such as amine than neutral or negatively charged ones. Since the pK a of OBP-1F is 5.20, the protein molecules are
Received 22 May 2016; Accepted 29 June 2016 DOI: 10.1002/pro.2976
Published online 4 July 2016 proteinscience.org
Abstract: The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA process- ing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.