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BBA - Proteins and Proteomics (v.1794, #9)
Know your limits: Assumptions, constraints and interpretation in systems biology
by Garth R. Ilsley; Nicholas M. Luscombe; Rolf Apweiler ⁎ (pp. 1280-1287).
Much of modern biological research can be organised under unifying concepts such as ‘Network Biology’ or ‘Systems Biology’. These provide frameworks for discussion and evaluation, which is particularly necessary given the large number of interconnected components being measured in the genomic era. Conversely, they embody simplifications and assumptions that place limits on what can be deduced from experimental data. Understanding these constraints is essential not only for scientific interpretation, but also in evaluating new experimental methods and conceptual advances.
Keywords: Systems biology; Functional genomics; Bioinformatics; Standards; Metadata
Structural organization of WrbA in apo- and holoprotein crystals
by Julie Wolfova; Ivana Kuta Smatanova; Jiri Brynda; Jeroen R. Mesters; Mikalai Lapkouski; Michal Kuty; Antonino Natalello; Neal Chatterjee; Sy-Yeu Chern; Erin Ebbel; Angela Ricci; Rita Grandori; Rüdiger Ettrich; Jannette Carey (pp. 1288-1298).
Two previously reported holoprotein crystal forms of the flavodoxin-like E. coli protein WrbA, diffracting to 2.6 and 2.0Å resolution, and new crystals of WrbA apoprotein diffracting to 1.85Å, are refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the protein's global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features.
Keywords: Twisted open-sheet fold; Electrostatic potential surface; Dimerization; Trichloroacetic acid; Disulfide; NAD(P)H:quinone oxidoreductase; Diffraction resolution
Structural overview on the allosteric activation of cyclic AMP receptor protein
by Hyung-Sik Won; Yoo-Sup Lee; Sung-Hee Lee; Bong-Jin Lee ⁎ (pp. 1299-1308).
Cyclic AMP receptor protein (CRP) is a prokaryotic global transcription regulator that controls the expression of nearly 200 genes. The protein, allosterically activated by cAMP binding, binds to DNA and interacts with RNA polymerase. Current understanding on the allosteric process of the Escherichia coli CRP activation can be summarized into a rigid-body movement that involves subunit realignment and domain rearrangement. The main consequence of that overall transition is protrusion and adjustment of F-helices that recognize specific DNA sites. Although physicochemical and structural studies during the past decades have contributed to a comprehensive understanding of the CRP allostery, a paucity of structural information about the cAMP-free form (apo-CRP) has precluded a definite elucidation of the allosterism. In this respect, recent achievements of structures on other CRP-family proteins provide useful information to fill in the details of the allosteric transition of CRP. Thus, in this paper, accomplishments of CRP-family structures are summarized and inspected comparatively with new findings. This review not only provides a structural overview on the allosteric conformational change of CRP but also suggests a thoughtful discussion about unsolved issues or conflicting arguments. Solving those issues and the apo-CRP structure would enable us to finally define the CRP allostery.
Keywords: Abbreviations; DBD; DNA-binding domain; NTD; N-terminal domain; apo-CRP; ligand-free CRP; cAMP–CRP; cAMP-bound CRP; CRP⁎; LA; constitutively active T127L/S128A mutant of CRP; Rr; CooA; ligand-free CooA from; Rhodospirillum rubrum; Ch; CooA; CooA from; Carboxydothermus hydrogenoformans; Ch; CooA⁎; LL; constitutively active N127L/S128A mutant of; Ch; CooA; Im-; Ch; CooA; imidazole-bound; Ch; CooA; Dd; CprK; reduced CprK from; Desulfitobacterium dehalogenans; Dh; CprK; ox; oxidized CprK from; Desulfitobacterium hafniense; CHPA; 3-chloro-4-hydroxyphenylacetate; CHPA-; Dh; CprK; ox; CHPA-bound; Dh; CprK; ox; PrfA⁎; 145; constitutively active G145S mutant of PrfA from; Listeria monocytogenes; NMR; nuclear magnetic resonance; SANS; small angle neutron scatteringAllostery; cAMP; CooA; CprK; CRP; PrfA
Cell-based proteome analysis: The first stage in the pipeline for biomarker discovery
by Gereon Poschmann; Barbara Sitek; Bence Sipos; Michael Hamacher; Oliver Vonend; Helmut E. Meyer; K. Stühler ⁎ (pp. 1309-1316).
The early detection of a distinct disease is crucial for a successful treatment and depends on a sensitive as well as specific diagnosis. In last years tremendous attempts were undertaken to identify new biomarker applying proteomics, but no relevant candidate has been identified for clinical application. Although proteomics is enabling quantitative and qualitative analysis of proteins within complex mixtures it could not significantly contribute to this field. Therefore, different proteomics approaches have been established focusing on the direct analysis of cell populations involved in pathogenic processes to identify candidate biomarkers even for in vitro diagnosis.Here, we will outline approaches applying cell- and tissue based proteome analysis as the first decisive step in the pipeline for the discovery of new diagnostic biomarkers. We will show examples for analysing precursor lesions of the pancreatic ductal adenocarcinoma (PDAC), nephron glomeruli and fibrotic and non-fibrotic liver tissue. This article provides also an overview about currently available techniques in the field of cell enrichment and quantitative proteome analysis of lowest amounts of sample.
Keywords: Abbreviations; CA; carrier ampholytes; FACS; fluorescence activated cell sorting; FFPE; formalin fixed paraffin embedded; GFP; green fluorescent protein; LCM; laser-capture microdissection; MACS; magnetic-activated cell sorting; MRN; multiple reaction monitoring; PDAC; pancreatic ductal adenocarcinoma; SOP; standard operating procedure; TMA; tissue microarray2D-DIGE; Biomarker; Differential proteome analysis; Microdissection; High-performance proteomic; Mass spectrometry
Hemoglobin, an “evergreen” red protein
by Stefano Bettati; Cristiano Viappiani; Andrea Mozzarelli ⁎ (pp. 1317-1324).
After more than a century of experimental, theoretical and computational studies, there is no general agreement yet on the mechanisms underlying the fine regulation of hemoglobin structural and functional properties. The experiments that we have carried out during the last two decades on hemoglobin immobilized in the crystal or in nanoporous silica gels have demonstrated that oxygen binding to a single quaternary structure is non-cooperative. This work finally settled the controversy among competing allosteric models. In addition, a vast amount of experimental evidence has been accumulated showing that tertiary conformational changes play a major role in the functional regulation of hemoglobin, even within a single quaternary state, an observation that is inconsistent with the classical MWC model. Experiments appear to be fully consistent with the Tertiary Two State allosteric model, recently proposed by Eaton and co-workers.The theoretical and experimental approaches described in this review should help in providing a quantitative understanding of allosteric interactions in other multi-subunit protein complexes.
Keywords: Abbreviations; CD; circular dichroism; Hb; hemoglobin; IHP; inositol hexaphosphate; MEM; maximum entropy method; MWC; Monod, Wyman and Changeux; PEG; polyethylene glycol; TTS; Tertiary Two State allosteric modelHemoglobin; Oxygen binding; Allostery; Microspectrophotometry; Laser flash photolysis; Crystal; Silica gel; Protein encapsulation
Towards understanding epithelial–mesenchymal transition: A proteomics perspective
by Rommel A. Mathias; Richard J. Simpson ⁎ (pp. 1325-1331).
Epithelial–mesenchymal transition (EMT) is recognised as a crucial embryonic process that converts immotile epithelial cells into migratory mesenchymal cells. More recently, EMT has been implicated in key stages of tumour progression, whereby it facilitates epithelial cancer cell metastasis. Traditionally, molecular and cell biology-based approaches have been utilised to study EMT. However, a recent wave of EMT research has implemented proteomic techniques to reveal new insights, and these integrated strategies have implicated novel proteins in the process of EMT. In this review, we highlight the important contributions proteomics has made in broadening our existing knowledge of EMT. Recent advances in sub-proteome sample preparation, proteomic tools including mass spectrometry instrumentation and protein quantitation, will further refine and extend our understanding of the cellular EMT process.
Keywords: Abbreviations; EMT; Epithelial–mesenchymal transition; MDCK; Madin–Darby canine kidney; EGF; Epidermal growth factor; TGF-β; Transforming growth factor-β; MS; Mass spectrometry; TCC; Transitional cell carcinoma; Maspin; Mammary serine protease inhibitor; ALCAM; Activated leukocyte cell adhesion molecule; MRM; Multiple reaction monitoring; 2-DE; Two-dimensional electrophoresis; DIGE; 2-D fluorescence difference gel electrophoresis; ECM; Extracellular matrix; iTRAQ; isobaric tag for relative and absolute quantitationEpithelial–mesenchymal transition; Proteomics; Orbitrap; Multiple reaction monitoring; Systems biology; Mass spectrometry
Protein secondary structure content in solution, films and tissues: Redundancy and complementarity of the information content in circular dichroism, transmission and ATR FTIR spectra
by Erik Goormaghtigh ⁎; Régis Gasper; Audrey Bénard; Andréa Goldsztein; Vincent Raussens (pp. 1332-1343).
The paper presents a simple and robust method to determine protein secondary structure from circular dichroism, transmission and attenuated total reflection (ATR) Fourier transform infrared spectra. It is found that the different spectroscopic methods bring valuable but roughly identical information on the secondary structure of proteins. ATR and transmission FTIR spectra display distinct differences, yet the secondary structure can be predicted from their spectra with roughly the same success. It is also found that one wavenumber or wavelength includes the large majority of the information correlated with secondary structure content and no more than 3 significant independent wavenumbers/wavelengths could be found for any of the spectroscopic data. This finding indicates that more complex linear combinations of the absorbance or ellipticities will not further improve secondary structure predictions. Furthermore, the information content in CD, transmission and ATR FTIR spectra is largely redundant. If combining CD and FTIR results in some improvement of structure prediction quality, the improvement is too modest to prompt spectroscopists to collect different spectroscopic data for structure prediction purposes. On the other hand, the data collected show that the quality of the FTIR spectrometers is such that biosensors or imaging methods sampling from 10−9 to 10−15 g yield spectra of sufficient quality to analyze protein secondary structure. These new techniques open the way to a new area of research, both in protein conformational response to ligand and imaging at sub-cellular scales.
Keywords: Secondary structure; IR spectroscopy; Circular dichroism; Protein film; FTIR imaging; FTIR sensors
Gly192 at hinge 2 site in the chaperonin GroEL plays a pivotal role in the dynamic apical domain movement that leads to GroES binding and efficient encapsulation of substrate proteins
by Kodai Machida; Ryoko Fujiwara; Tatsuhide Tanaka; Isao Sakane; Kunihiro Hongo; Tomohiro Mizobata; Yasushi Kawata ⁎ (pp. 1344-1354).
The subunit structure of chaperonin GroEL is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The ‘hinge 2’ site of GroEL contains three glycine residues, Gly192, Gly374, and Gly375, connecting the apical domain and the intermediate domain. In this study, to understand the importance of the hinge 2 amino acid residues in chaperonin function, we substituted each of these three glycine residues to tryptophan. The GroEL mutants G374W and G375W were functionally similar to wild-type GroEL. However, GroEL G192W showed a significant decrease in the ability to assist the refolding of stringent substrate proteins. Interestingly, from biochemical assays and characterization using surface plasmon resonance analysis, we found that GroEL G192W was capable of binding GroES even in the absence of ATP to form a very stable GroEL–GroES complex, which could not be dissociated even upon addition of ATP. Electron micrographs showed that GroEL G192W intrinsically formed an asymmetric double ring structure with one ring locked in the ‘open’ conformation, and it is postulated that GroES binds to this open ring in the absence of ATP. Trans-binding of both substrate protein and GroES was observed for this binary complex, but simultaneous binding of both substrate and GroES (a mechanism that ensures substrate encapsulation) was impaired. We postulate that alteration of Gly192 severely compromises an essential movement that allows efficient encapsulation of unfolded protein intermediates.
Keywords: Abbreviations; CD; circular dichroism; Gdn–HCl; guanidine hydrochloride; GFP; green fluorescent protein; G; nnn; W; glycine; →; tryptophan substituted GroEL mutant at; nnn; position; WT; wild-type chaperonin GroEL; SR1; single ring mutant of wild-type GroELChaperonin; GroEL; Hinge 2 site; Structure and function; Protein folding; GroEL–GroES complex formation
Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease
by Jessica Patterson-Ward; Johnathan Tedesco; Jason Hudak; Jennifer Fishovitz; James Becker; Hilary Frase; Kirsten McNamara; Irene Lee ⁎ (pp. 1355-1363).
Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate λN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the kcat of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of λN and truncated λN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout λN to achieve substrate promiscuity.
Keywords: Abbreviations; Abz; anthranilamide; ATP; adenosine tri-phosphate; ADP; adenosine di-phosphate; AMPPNP; adenylyl 5-imidodiphosphate; Boc; tert-butoxycarbonyl; BSA; bovine serum albumin; Bz; benzoic acid amide; Dansyl; 5-(dimethylamino)naphthalene-1-sulfonyl; Dlu; density light units; DTT; dithiothreitol; Fmoc; 9; H; -fluoren-9-ylmethoxycarbonyl; FRET; fluorescence resonance energy transfer; HBTU; O-benzotriazole-; N; ,; N; ,; N; ′,; N; ′-tetramethyluroniumhexafluorophosphate; HEPES; N; -2-hydroxyethylpiperzaine-; N; ′-ethanesulphonic acid; IPTG; isopropyl-beta-D-thiogalactopyranoside; KOAc; potassium acetate; λN; also known as the lambda N protein, a lambda phage protein that allows; E. coli; RNA polymerase to transcribe through termination signals in the early operons of the phage; Mg(OAc); 2; magnesium acetate; NO; 2; nitro; PEI-cellulose; polyethyleneimine-cellulose; SDS; sodium dodecyl sulfate; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TLC; thin layer chromatography; TPCK; tosyl phenylalanyl chloromethyl ketone; Tris; 2-amino-2-hydroxymethyl-1,2-propanediolATP-dependent protease; Fluorogenic peptidase assay; Mechanistic probe
Tracing metabolic pathways from enzyme data
by Andrew G. McDonald; Keith F. Tipton; Sinéad Boyce (pp. 1364-1371).
The IUBMB Enzyme List is widely used by other databases as a source for avoiding ambiguity in the recognition of enzymes as catalytic entities. However, it was never designed for activities such as pathway tracing, which have become increasingly important in systems biology. This is because it often relies on generic or representative reactions to show the reactions catalysed by enzymes of wide specificity. It is necessary to go to databases such as BRENDA to find further, more detailed, information on what is known about the range of substrates for any particular enzyme. In order to provide a framework for tracing pathways involving any specific enzyme or metabolite, we have created a Reactions Database from the material in the Enzyme List. This allows reactions to be searched by substrate/product and pathways to be traced from any selected starting/seed substrate. An extensive synonym glossary allows searches by many of the alternative names, including accepted abbreviations, by which a chemical compound may be known. This database was necessary for the development of the application Reaction Explorer (http://www.reaction-explorer.org), which was written in REALbasic to search the Reactions Database and draw metabolic pathways from reactions selected by the user. Having input the name of the starting compound (the “seed”), the user is presented with a list of all reactions containing that compound and then selects the product of interest as the next point on the ensuing graph. The pathway diagram is then generated as the process iterates. A contextual menu is provided, which allows the user to (i) remove a compound from the graph, along with all associated links; (ii) search the reactions database again for additional reactions involving the compound and (iii) search for the compound within the Enzyme List.
Keywords: Database; Enzyme; Metabolic pathway
Cysteine cathepsins are not critical for TNF-α-induced cell death in T98G and U937 cells
by Martina Klarić; Sun Tao; Veronika Stoka; Boris Turk; Vito Turk ⁎ (pp. 1372-1377).
The tumor necrosis factor (TNF) is a cytokine known to be an important mediator of apoptosis and inflammation. It has been implicated in the pathogenesis of a number of diseases, including cancer and rheumatoid arthritis. TNF apoptosis has been known for a number of years to be critically dependent on caspases; however, recently it has been suggested that cysteine cathepsins might also be involved in the pathway. In the present work the hypothesis that cathepsins can act as an essential downstream mediator of TNF-α-triggered apoptosis was tested. The TNF-α apoptosis was investigated in two tumor-cell lines: U937 and T98G. Based on the use of pharmacological caspase inhibitors, the TNF-α induced caspase-dependent apoptotic cell death in both cell lines, which was accompanied by lysosomal destabilization and the release of cathepsins in the cytosol. However, blocking cysteine cathepsins with a broad-spectrum inhibitor, E64d, or a more specific cathepsin B inhibitor, CA-074Me, had no effect on the progression of the apoptosis in both cell lines, suggesting that the TNF-α apoptosis is not critically dependent on the cathepsins in these two cellular models.
Keywords: Abbreviations; CA-074Me; [(2S,3S)-3-Propylcarbamoyloxirane-2-carbonyl]-; l; -isoleucyl-; l; -proline methyl ester; CHX; cycloheximide; E-64d; (2; S; ,3; S; )-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester; TNF-α; tumor necrosis factor-alpha; Z-DEVD-FMK; Z-Asp(OMe)-Glu(OMe)-Val-; dl; -Asp(OMe)-fluoromethylketone; Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketoneTNF-α; Apoptosis; Cathepsin B; Cathepsin; Caspase; Cysteine protease
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