Phosphorylation in Yeast.

Background: -Phosphorylation is the addition of a orthophosphate (PO4) group to a protein or another(prenominal)(prenominal) organic molecule. Protein phosphorylation in particular plays a meaning(prenominal) place in a wide grasp of cellular carry break throughes. In Biochemistry protein phosphorylation has made a signifi quite a littlet contribution in researches throughout una manage biological fields. If we carriage at the front researches, in 1906 Phoebus Levene at Rockefeller institute, place phosphate in the protein Vitellin and after(prenominal) that in 1933 he detected phosphoserine in Casein, a milk protein. But it wasn?t until twenty eld originally Eugene Kennedy exposit the beginning ?enzymatic phosphorylation of proteins?. Phosphorylation of proteins is an important restrictive machine which occurs in both prokaryotes and eukaryotes. Enzymes called kinases, which atomic fall 18 apply in phosphorylation, atomic number 18 tough in this process. Dur ing the process both(prenominal) enzymes and receptors ar switched ?on? or ?off? by phosphorylation and dephosphorylation. Reversible protein phosphorylation provides a major(ip) regulatory mechanism in eukaryotic cells. Due to broad(prenominal) variant of amino vinegarish resi due(p)s producing a relatively limited get of experimentally identified phosphorylation sites, on that point is no reliable mark of much(prenominal) sites. Phosphorylation usually occurs on serine, threonine and tyrosine residues in eukaryotic proteins. more everywhere, in prokaryotic proteins phosphorylation takes place on sancti iodind amino venereal infection residues such as histidine, arginine or lysine. Protein phosphorylation catalysed by protein kinases plays a major role in cellular sign of the zodiac. on that point be thousands of unequivocal phosphorylation sites in a given cell because there are galore(postnominal) varied kinds of proteins in any cell. It has been estimated that 1/tenth to ½ of proteins are phosphor! ylated. Phosphorylation occurs mostly on multiple discrete sites on a given protein. Phosphorylation of any site on a given protein move change function of jam of that protein. at heart a protein, phosphorylation support occur on several amino acids. Phosphorylation on serine is most common, followed by threonine.. Protein kinases execution on and alter the activity of item proteins. These are utilize extensively to transplant signals and control complex processes in cells. Up to 500 different kinases find been identified in humans. A typical eukaryotic cell has the capacity to make surface 30,000 different proteins, which catalyse thousands of different reactions involving many metabolites. Cyclin-dependent kinases (CDK) belong to a group of protein kinases primitively discovered as being compound in the ordination of the cell cycle. CDKs phosphorylate proteins on serine and threonine amino acid residues: they are serine/threonine kinases. A cyclin-dependent kinase i s activated by association with a cyclin, forming a cyclin-dependent kinase complex. maven of the experimental conditioninuss to understand which is utilize in the word is a ? term motif?, which is a stem or an amino acid sequence pattern that is general and has, or is conjectured to oblige a biological significance. Within a sequence or entropybase of sequences, researchers search and find motifs victimization computer-based techniques of sequence analysis, such as BLAST. Bioinformatics field operates to use such techniques. The new engineering in market these days is the protein- cow dung applied acquisition which allows thorough analysis of biochemical activities and can be apply to analyze protein kinases as well. Protein micro sets devising use of new developments in protein engineering and detection stick out recently emerged. The basic construction of such protein chips has roughly similarities to deoxyribonucleic acid chips, such as the use of glass or ductile surface continue with an array of molecu! les. With fluorescent markers or other methods of detection telling the spots that score captured these proteins, protein micro arrays are being utilize as powerful tools in game-throughput proteomics and dose uncovering. At this time, protein-chip engineering concentrates on understanding molecular pathways that in turn triad to useful information for drug discovery and which can be applied to molecular diagnostics. CRITIQUE: -Realizing the surfaceableness of protein phosphorylation which controls many cellular processes, the authors of this article have identified over 4000 phosphorylation events which involved over a thousand proteins. They used the proteome-chip engineering science to describe the in vitro substrates know by the protein kinases. The authors believe that the results go away shed clear(p) on mechanisms and roles of protein phosphorylation in eukaryotes. This is understood as protein phosphorylation controls many processes in the cells. Moreover barms possess many protein kinases and avail as comfortably organisms for study. The authors study different protein kinases along with A ones which are a family of enzymes whose activity is dependent on the train of cyclic AMP. Protein kinase A has several functions in cell like glycogen and lipide metabolism. A brief note about the protein kinases would have been useful. They then carried out proteome micro arrays on the kinases by using 33-P tagged isotope on ATP. The authors can be commended no their use of shun and positive controls of the experiment. They made the experiment easier by using a freehanded number of proteins and sources for identification of phosphorylation signals. They as well as used incubated slides in absence of kinases so as to pass along if any autophosphorylated. This served as a blank. They measured the extent of phosphorylation by algorithmic methods. Although this method whitethorn be 70 to 80 percent (according to various sources) effective, it is pertinent here as many kinases need to be analyz! ed and the method is widely sanctioned in this era of technology. The phosphorylated proteins were marked as positive substrates in the results. According to the results, since many phosphorylation events occurred, the authors said that separately kinase had a certain(p) sum total (between 1 and 256) of substrates with an average of forty seven substrates per enzyme. nevertheless though criticizing the authors for not giving accurate results would be incorrect, mean heretofore can be made that an average as such cannot be taken which has a standard aside so high but for the information of reader it can be accepted. What this means is that an average for kinase activity would not be relevant in this case. The authors mentioned that more than one kinases had about ternary substrates indicating specificity. There may be an element of doubt in this matter. Enzymes are highly specific molecules. There may have been the like kinase they analyzed but due to improper labeling, gav e different results. It is nonetheless manageable that the enzymes have more than one substrate. The term ? tuneful arrangement computes were the largest proteins phosphorylated? was not in force(p)y understood. They ground that the kinases phosphorylated the proteins which resided in the aforementioned(prenominal) cell compartment or which came under the same function and this was hear with an example. The authors then carried out a test in which they assayed substrates of important kinases to test various other factors. This complex test gave some unattackable results as would be expected. This proved that the kinases controlled protein levels directly or indirectly. The went shape up to say that using the proteome micro array technology helped them identify many genuine substrates and proved that phosphorylation alter protein levels with a marked difference. Next, they examined the kinases which were closely related to a substrate. They used yeast protein kinase A h omologues. They went on to determine if these homolog! ues were operationally redundant. From results they cogitate that each homologue verified specificity. More than 3/4th of bovid cAMP-dependent protein kinase physical objects were withal substrates of the homologues. The slides with 90% substrate point of carrefour depicted that each homologue had distinct substrate specificity. A good diagram gave information on how the proteome array test was carried out by in vivo institution. enter 2(a) showed that TPK1 had highest substrate recognition visibleness. numeral 2(b) showed how cyclin affected substrate recognition. It was in like manner found that position and coordinate of the substrate restricted the kinase from phosphorylation. Later, the 4,200 proteins-substrate phosphorylations were assembled into an in vitro intercommunicate to be analyzed and a mapping was seduced. From this they found that identification of substrate support in defining the role of kinase in yeast signaling networks. They said the low overlap between the data sets was due to kinase-substrate interaction had lo bandaging affinities and were not detectable in the assay. waiver in depth with the phosphorylation network, in which the kinase and substrate were present in the same compartment or functional category, resulted in 33% and 18.4% interactions, respectively. They sketch a limitation to this onrush of enrichment saying that this advance may give false negatives if the data interpreted were inaccurate or incomplete. The figure of in vivo phosphorylation map given was similar to that of the yeast, genus Saccharomyces cerevisiae.

then after, to explain how phosphorylation is combine in global networks, they have phosph orylation data with recording factor binding to gene! rate an incorporated regulatory network for yeast. arrangement factor is a protein that binds to specific sequence of DNA and t hence controls the transfer of heritable information from DNA to RNA. The number of transcription factors which can be found in an organism increases with the genome size and the larger genomes tend to have more transcription factors per gene. Then , within the network they looked for common regulatory modules. Eight were discover from which six were of high importance statistically. The authors think their study is the first global investigation of protein phosphorylation by protein kinases using an unbiased approach. In the article, the term scaffold refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding divers(a) target molecules with antibody-like properties of specificity and affinity. The authors have also studied antecedent in vivo phosphorylations, so that would give them an excess edge on the return they pursued. When trust their data with others, it matched their studies as well. Even though the authors flattery proteome chip technology, it has its side personal effects but on large scale protein purge, it has been proven effective. boundary of proteome chips:An important obstacle in fabricating proteome chips is the need to scram and throw up a large number of proteins in a punctual manner. In recent years there has been rapid advancement in improving the throughput process and implementing alternative approaches. In vitro transcription-translation systems hold out an alternative approach for producing proteins. Scientists have taken this process one step further by directly applying in vitro transcription-translation systems to protein chip fabrication. The obvious advantage is that the dear(predicate) and time-consuming steps of ORF cloning, protein expression and purification can be eliminated. However, because of inherent problems with the in vitro system, the size of proteins that can be produced is ! limited, and the reproducibility of the system is not high. One possible limitation of this approach is that problems with protein folding and the integrity of the proteins printed on the chips aptitude sometimes produce false-positives or missed points. However, the possibility of using a range of different DNA probes, each with a specific modification, to profile cellular proteins on the whole-proteome level is a good prospect. If, as shown here, one or two proteins can be identified with each probe that is applied, a whole database can be built for further biological validation and reference. In the article, the term scaffold refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The authors have also studied previous in vivo phosphorylations so that would give them an extra edge on the topic they pursued. When combining their data with othe rs, it matched their studies as well. Conclusion: -Overall, the authors did an smooth job in the analysis of phosphorylation of yeast. They used the latest technology available to create algorithms and networks which helped in analyzing the substrate-kinase relationships. The proteome chips allows rapid and automated resoluteness to protein phosphorylation, saves time and is efficient than other out-dated techniques. The article also gives an cortical potential into what could be further done to take this topic to another level for research, and hence can lay a derriere to it. References: -Global analysis of protein phosphorylation in yeast: Jason?Snyder, 2005 nature publishing group. Nelson and Cox, Lehninger principles of biochemistry, fifth edition:2008Koch C, et al. mobile phone cycle regulated transcription in yeast. 1994 Jun;6(3):451-9. Nasmyth K. Control of the yeast cell cycle by the Cdc28 protein kinase. 1993 Apr; 5(2):166-79. hypertext transfer protocol://www.deviceli nk.com/ivdt/archive/02/07/002.htmlhttp://www.hosted-w! ebs.com/phosphorylation/http://en.wikipedia.org/wiki/Phosphorylationhttp://www.jbc.org/cgi/content/abstract/252/9/3082http://biowww.net/detail-1142.htmlhttp://en.wikipedia.org/wiki/AMP-activated_protein_kinasehttp://www.rockefeller.edu/labheads/crossf/cdk.htmlhttp://networks.gersteinlab.org/http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2576280http://www.functionalgenomics.org.uk/sections/resources/protein_arrays.htmhttp://genomebiology.com/2001/2/10/reports/0035 If you want to get a full essay, order it on our website: OrderEssay.net

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