Misplaced Pages

#164835

35-434: P-bodies were originally proposed to be the sites of mRNA degradation in the cell and involved in decapping and digestion of mRNAs earmarked for destruction. Later work called this into question suggesting P bodies store mRNA until needed for translation. In neurons , P-bodies are moved by motor proteins in response to stimulation. This is likely tied to local translation in dendrites . P-bodies were first described in

70-427: A recombinant form of streptavidin with a near-neutral pI is also commercially available. Pretargeted immunotherapy uses streptavidin conjugated to a monoclonal antibody against cancer cell-specific antigens followed by an injection of radiolabelled biotin to deliver the radiation only to the cancerous cell. Initial hurdles involve saturation of the biotin binding sites on streptavidin with endogenous biotin instead of

105-472: A covalent linkage occurs to enable higher number of biotin binding sites. Six and twelve biotin binding sites per molecule have been made with this method. Streptavidin is not the only protein capable of binding to biotin with high affinity. Avidin is the other most notable biotin-binding protein. Originally isolated from egg yolk, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. Avidin has

140-415: A functional biotin binding site and purification by ion-exchange chromatography . The functional binding sites here have the same biotin binding stability as wild-type streptavidin. Divalent streptavidin with the two biotin binding sites together (cis-divalent) or apart (trans-divalent) can be separately purified. A streptavidin with exactly three biotin binding sites per tetramer can also be produced using

175-437: A higher affinity for biotin ( K d ~ 10 M) but in contrast to streptavidin, avidin is glycosylated, positively charged, has pseudo-catalytic activity (avidin can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation. On the other hand, streptavidin is the better biotin-conjugate binder; avidin has a lower binding affinity than streptavidin when biotin

210-446: A hub around which other proteins may be arranged, either by an affinity tag such as Strep-tag or AviTag or by genetic fusion to SpyTag . Fusion to SpyTag allowed generation of assemblies with 8 or 20 streptavidin subunits. As well as a molecular force probe for atomic force microscopy studies, novel materials such as 3D crystalline lattices have also been created. Streptavidin has a mildly acidic isoelectric point (pI) of ~5, but

245-520: A lowered biotin-binding affinity, which is to be expected in such a highly optimized system. However, a recently engineered mutant of streptavidin, named traptavidin, was found to have more than ten-fold slower biotin dissociation, in addition to higher thermal and mechanical stability. This decreased dissociation rate was accompanied by a two-fold decrease in the association rate. Biotin-binding affinity can be impaired by chemical labeling of streptavidin, such as with amine-reactive fluorophores ; flavidin

280-589: A novel site termed EGP bodies, or stress granules, may be responsible for mRNA storage as these sites lack the decapping enzyme. microRNA mediated repression occurs in two ways, either by translational repression or stimulating mRNA decay. miRNA recruit the RISC complex to the mRNA to which they are bound. The link to P-bodies comes by the fact that many, if not most, of the proteins necessary for miRNA gene silencing are localized to P-bodies, as reviewed by Kulkarni et al. (2010). These proteins include, but are not limited to,

315-516: Is a context dependent (stress state versus normal) specificity to the P-body's mechanism of action. Based on the evidence that P-bodies sometimes are the site of mRNA decay and sometimes the mRNA can exit the P-bodies and re-initiate translation, the question remains of what controls this switch. Another ambiguous point to be addressed is whether the proteins that localize to P-bodies are actively functioning in

350-453: Is a streptavidin mutant without lysine side-chains, which retains good biotin binding characteristics after such fluorescent dye labeling where the dye couples to the amino terminus. Among the most common uses of streptavidin are the purification or detection of various biomolecules. The strong streptavidin-biotin interaction can be used to attach various biomolecules to one another or onto a solid support. Harsh conditions are needed to break

385-450: Is a tetramer but only one of the four binding sites is functional. This single binding site has 10 mol/L affinity and cannot cause cross-linking. Applications of monovalent streptavidin have included fluorescent tracking of cell surface receptors , decorating DNA origami , and acting as a pointer to identify specific regions for cryo-electron microscopy . Monomeric streptavidin is a recombinant form of streptavidin with mutations to break

SECTION 10

#1732858564165

420-433: Is also thought to account for the high affinity. In particular, the pocket is lined with conserved tryptophan residues. Lastly, biotin binding is accompanied by the stabilisation of a flexible loop connecting β-strands 3 and 4 (L3/4), which closes over the bound biotin, acting like a 'lid' over the binding pocket and contributing to the extremely slow biotin dissociation rate. Most attempts at mutating streptavidin result in

455-408: Is an advantage in applications like MHC tetramer staining , where avidity effects improve the ability of MHC molecules attached to streptavidin to detect specific T cells. In other cases, such as the use of streptavidin for imaging specific proteins on cells, multivalency can perturb the function of the protein of interest. Monovalent streptavidin is an engineered recombinant form of streptavidin which

490-459: The 5' cap structure on the RNA exposing a 5' monophosphate. In eukaryotes , this 5' monophosphate is a substrate for the 5' exonuclease Xrn1 and the mRNA is quickly destroyed. There are many situations which may lead to the removal of the cap, some of which are discussed below. In prokaryotes , the initial mRNA transcript naturally possesses a 5'-triphosphate group after bacterial transcription ;

525-676: The Strep-tag peptide. Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase . Streptavidin has also been used in the developing field of Nanobiotechnology , the use of biological molecules such as proteins or lipids to create nanoscale devices/structures. In this context streptavidin can be used as a building block to link biotinylated DNA molecules to create single walled carbon nanotube scaffolds or even complex DNA polyhedra. The tetrameric streptavidin has also been used as

560-577: The exosome . Certain classes of miRNA have also been shown to stimulate decapping. Streptavidin Streptavidin / ˌ s t r ɛ p ˈ t æ v ɪ d ɪ n / is a 52 kDa protein (tetramer) purified from the bacterium Streptomyces avidinii . Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (K d ) on

595-638: The 159 residue full-length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13–139; removal of the N and C termini is necessary for the highest biotin-binding affinity. The secondary structure of a streptavidin monomer is composed of eight antiparallel β-strands, which fold to give an antiparallel β-barrel tertiary structure. A biotin binding-site is located at one end of each β-barrel. Four identical streptavidin monomers (i.e. four identical β-barrels) associate to give streptavidin's tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from

630-530: The BirA* enzyme. When the cells are incubated with biotin , BirA* will biotinylate proteins that are nearby, thus tagging the proteins within processing bodies with a biotin tag. Streptavidin was then used to isolate the tagged proteins and mass spectrometry to identify them. Using this approach, Youn et al. identified 42 proteins that localize to processing bodies. Messenger RNA decapping The process of messenger RNA decapping consists of hydrolysis of

665-432: The binding pocket and biotin. Secondly, there is an extensive network of hydrogen bonds formed to biotin when in the binding site. There are eight hydrogen bonds directly made to residues in the binding site (the so-called 'first shell' of hydrogen bonding), involving residues Asn23, Tyr43, Ser27, Ser45, Asn49, Ser88, Thr90 and Asp128. There is also a 'second shell' of hydrogen bonding involving residues that interact with

700-500: The cap by the decapping enzyme DCP2 and protects the mRNA molecule. In nutrient-starvation conditions or viral infection, translation may be compromised and decapping is stimulated. This balance is reflected in the size and abundance of the cytoplasmic structures known as P-bodies . A number of specific decay pathways exist that recognize aberrant messages and promote their decapping. Nonsense mediated decay recognizes premature stop codons and promotes decapping as well as decay by

735-431: The enzyme RppH removes a pyrophosphate molecule from the 5' end, converting the 5'-triphosphate to a 5'-monophosphate, triggering mRNA degradation by ribonucleases. Inside cells, there is a balance between the processes of translation and mRNA decay. Messages which are being actively translated are bound by polysomes and the eukaryotic initiation factors eIF-4E and eIF-4G (in eukaryotes). This blocks access to

SECTION 20

#1732858564165

770-399: The first shell residues. However, the streptavidin-biotin affinity exceeds that which would be predicted from the hydrogen bonding interactions alone, suggesting another mechanism contributing to the high affinity. The biotin-binding pocket is hydrophobic , and there are numerous van der Waals force -mediated contacts and hydrophobic interactions made to the biotin when in the pocket, which

805-497: The injected radiolabelled biotin, and a high degree of radioactive exposure in the kidneys due to streptavidin's strong cell adsorptive properties. It is currently thought that this high level of binding to adherent cell types, such as activated platelets and melanomas, is a result of integrin binding mediated through the RYD sequence in streptavidin. Streptavidin is a tetramer and each subunit binds biotin with equal affinity. Multivalency

840-430: The interior of the barrel, together with a conserved Trp120 from a neighboring subunit. In this way, each subunit contributes to the binding site on the neighboring subunit, and so the tetramer can also be considered a dimer of functional dimers. The numerous crystal structures of the streptavidin-biotin complex have shed light on the origins of the remarkable affinity. Firstly, there is high shape-complementarity between

875-636: The miRNA gene silencing process or whether they are merely on standby. In 2017, a new method to purify processing bodies was published. Hubstenberger et al. used fluorescence-activated particle sorting (a method based on the ideas of fluorescence-activated cell sorting ) to purify processing bodies from human epithelial cells. From these purified processing bodies they were able to use mass spectrometry and RNA sequencing to determine which proteins and RNAs are found in processing bodies, respectively. This study identified 125 proteins that are significantly associated with processing bodies. Notably this work provided

910-547: The most compelling evidence up to this date that P-bodies might not be the sites of degradation in the cell and instead used for storage of translationally repressed mRNA. This observation was further supported by single molecule imaging of mRNA by the Chao group in 2017. In 2018, Youn et al. took a proximity labeling approach called BioID to identify and predict the processing body proteome. They engineered cells to express several processing body-localized proteins as fusion proteins with

945-479: The order of ≈10 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride ), detergents (e.g. SDS , Triton X-100 ), proteolytic enzymes, and extremes of temperature and pH. The crystal structure of streptavidin with biotin bound

980-459: The same principle as to produce divalent streptavidins. Streptavidins of higher valency has been obtained by utilizing the chemistry of isopeptide bond conjugation using the SpyTag/SpyCatcher technology. This involves having a streptavidin tetramer with three biotin binding sites and a dead streptavidin fused to either SpyTag or SpyCatcher. When the different tetramers are mixed together,

1015-459: The scaffold protein GW182, Argonaute (Ago), decapping enzymes and RNA helicases . The current evidence points toward P-bodies as being scaffolding centers of miRNA function, especially due to the evidence that a knock down of GW182 disrupts P-body formation. However, there remain many unanswered questions about P-bodies and their relationship to miRNA activity. Specifically, it is unknown whether there

1050-419: The scientific literature by Bashkirov et al. in 1997, in which they describe "small granules… discrete, prominent foci" as the cytoplasmic location of the mouse exoribonuclease mXrn1p. It wasn’t until 2002 that a glimpse into the nature and importance of these cytoplasmic foci was published., when researchers demonstrated that multiple proteins involved with mRNA degradation localize to the foci. Their importance

1085-566: The scientific literature. Recently evidence has been presented suggesting that GW-bodies and P-bodies may in fact be different cellular components. The evidence being that GW182 and Ago2, both associated with miRNA gene silencing, are found exclusively in multivesicular bodies or GW-bodies and are not localized to P-bodies. Also of note, P-bodies are not equivalent to stress granules and they contain largely non-overlapping proteins. The two structures support overlapping cellular functions but generally occur under different stimuli. Hoyle et al. suggests

P-bodies - Misplaced Pages Continue

1120-443: The streptavidin-biotin interaction, which often denatures the protein of interest being purified. However, it has been shown that a short incubation in water above 70 °C will reversibly break the interaction (at least for biotinylated DNA) without denaturing streptavidin, allowing re-use of the streptavidin solid support. A further application of streptavidin is for purification and detection of proteins genetically modified with

1155-409: The tetramer into a monomer and to enhance the solubility of the resultant isolated subunit. Monomeric streptavidin versions have an affinity for biotin of 10 mol/L 10 mol/L and so are not ideal for labeling applications but are useful for purification, where reversibility is desirable. A streptavidin with exactly two biotin binding sites per tetramer can be produced by mixing subunits with and without

1190-409: Was recognized after experimental evidence was obtained pointing to P-bodies as the sites of mRNA degradation in the cell. The researchers named these structures processing bodies or "P bodies". During this time, many descriptive names were used also to identify the processing bodies, including "GW-bodies" and "decapping-bodies"; however "P-bodies" was the term chosen and is now widely used and accepted in

1225-479: Was reported by two groups in 1989. The structure was solved using multi wavelength anomalous diffraction by Hendrickson et al. at Columbia University and using multiple isomorphous replacement by Weber et al. at E. I. DuPont Central Research and Development Department. As of September 2017, there are 171 structures deposited in the Protein Data Bank . See this link for a complete list. The N and C termini of

#164835