Cellular Protein Factory Self Assembly

C O N T E N T S:

KEY TOPICS

  • Self assembly follows a definite sequence. ie is not just 34 proteins coming together at random and somehow getting it right.(More…)
  • Monitoring the dynamics of thousands of protein complexes simultaneously within intact cells PHYS.ORG Protein-protein interactions are at the heart of all cellular functions and biological processes.(More…)
  • A recent model proposed that TAV is involved in the intracellular movement of CaMV factories, since EDIBs were observed near plasmodesmata and TAV interacts with CaMV movement protein P1, PDLP1 (Plasmodesmatal-Located Protein 1) and AtSRC2 (Soybean Response to Cold), two cellular proteins localized next to the tubules formed by P1.(More…)
  • Ribosomes bound to the ER of the host cell make the protein parts of the envelope glycoproteins; cellular enzymes in the ER and Golgi the add the sugars.(More…)

POSSIBLY USEFUL

  • Ribosomes, the molecular factories that produce all the proteins a cell needs to grow and function, are themselves made up of many different proteins and four RNAs.(More…)
  • FIG 8 Intracellular localization of AAP proteins of 10 different AAV serotypes and correlation with VP expression and capsid formation. (A) Expression plasmids encoding AAPs of the AAV serotypes shown were transfected together with a corresponding AAP-deficient helper plasmid into HeLa cells.(More…)

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Cellular Protein Factory Self Assembly
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link: http://science.sciencemag.org/content/355/6331/eaam5488.full
author: science.sciencemag.org
description: Self-assembly of genetically encoded DNA-protein hybrid nanoscale …

KEY TOPICS

Self assembly follows a definite sequence. ie is not just 34 proteins coming together at random and somehow getting it right. [1] In cells infected by some animal viruses, viroplasms result from the assembly of small aggregates that are transported by dynein along microtubules to the microtubule organization centre at the periphery of the nucleus, where they recruit cellular proteins and mitochondria. [2] IDPs have emerged as a major class of proteins that are involved in diverse cellular processes ranging from stress response to the regulation of organelle assembly, RNA availability, and transcription. [3]

Monitoring the dynamics of thousands of protein complexes simultaneously within intact cells PHYS.ORG Protein-protein interactions are at the heart of all cellular functions and biological processes. [4] This supports the idea that AAP directly interacts with AAV capsid proteins, rather than translocating VP to specific cellular structures for interaction with other factors. [5]

Results from such imaging studies using live cells suggest that the interaction frequency detected by 3C assays more likely reflect a complex convolution of physical proximity, DNA looping frequency, local chromatin environment, and cellular heterogeneity ( Fudenberg and Imakaev, 2017 ). [6]

The discovery that the serotype-specific AAPs differ in their activity ( Fig. 5 ) encouraged us to study whether they also display unique cellular localization patterns, perhaps distinct from the nucleolar signals reported for AAP2 ( 17 ). [5] Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. [5] Protein-protein interactions are at the heart of all cellular functions and biological processes. [4]

There isn?t a factory in the cell, that puts ribosomes together. [1] Insect cells as a factory to produce adeno-associated virus type 2 vectors. [5]

What you are asking about is “self assembly” of ribosomes which was demonstrated by Masayasu Nomura in the late 1960’s and early 1970’s. [1] The idea with self assembly, is the molecules “try” thousands of different associations. [1]

A recent model proposed that TAV is involved in the intracellular movement of CaMV factories, since EDIBs were observed near plasmodesmata and TAV interacts with CaMV movement protein P1, PDLP1 (Plasmodesmatal-Located Protein 1) and AtSRC2 (Soybean Response to Cold), two cellular proteins localized next to the tubules formed by P1. [2] Campagna M, Marcos-Villar L, Arnoldi F, de la Cruz-Herrera CF, Gallego P, Gonzalez-Santamaria J, et al. Rotavirus viroplasm proteins interact with the cellular SUMOylation system: implications for viroplasm-like structure formation. [2] K. D. Collins, “Ion hydration: Implications for cellular function, polyelectrolytes, and protein crystallization,” Biophysical Chemistry, vol. 119, no. 3, pp. 271-281, 2006. [7]

Since the C-terminal part of D1 is also involved in the RNA silencing suppression activity of TAV, this activity may contribute to the expression of symptoms in CaMV-infected plants by modifying the expression pattern of cellular genes as observed in transgenic Arabidopsis plants expressing TAV. [2] Their cellular localization is due to the presence of a NES (in domain A) and two NLSs (in the downstream region) that are involved in TAV nucleo-cytoplasmic shuttling ( Fig 1 ). [2]

Merge images showing the cellular distribution of EDIBs and ELIB, and TAVm3 and P2 bodies are presented in A, panel 3, and B, panel 3. [2]

Ribosomes bound to the ER of the host cell make the protein parts of the envelope glycoproteins; cellular enzymes in the ER and Golgi the add the sugars. [8]

POSSIBLY USEFUL

Ribosomes, the molecular factories that produce all the proteins a cell needs to grow and function, are themselves made up of many different proteins and four RNAs. [9] Ribosomes are the “protein factories” of the cell, and are composed of two units: a larger 50S subunit, and a smaller 30S subunit. [9] Ribosomes are the organelles responsible for protein synthesis in cells. [9]

Observing the cell’s protein factories during self-assembly: Electron microscopy images provide important insights into ribosome assembly. [10] These could potentially inhibit ribosome assembly, thereby paralyzing the bacterium’s ability to synthesize proteins; these drugs could therefore be used to inhibit all bacterial growth. [10]

A ribosome, in turn, is built from proteins and ribosomal RNAs stitched together with immaculate precision. [9] “Observing the cell’s protein factories during self-assembly: Electron microscopy images provide important insights into ribosome assembly.” [10] All of the proteins necessary for life are made by giant molecular machines called ribosomes. [9]

The lab of Federica Brandizzi is showing how a gene helps plants adjust protein production during stressful times. [9] The 50S subunit in turn comprises 33 proteins and two ribonucleic acid molecules. [9] July 19, 2017 – A batch of single-cell protein has been produced by using electricity and carbon dioxide, report scientists. [10]

Using 3-D cryo-electron microscopy, they were able to produce high-resolution snapshots of individual stages of the 50S subunit’s assembly and maturation. [9]

FIG 8 Intracellular localization of AAP proteins of 10 different AAV serotypes and correlation with VP expression and capsid formation. (A) Expression plasmids encoding AAPs of the AAV serotypes shown were transfected together with a corresponding AAP-deficient helper plasmid into HeLa cells. [5] We therefore triple transfected human embryonic HEK293T cells with a green fluorescent protein (GFP)-encoding self-complementary AAV vector plasmid, an adenoviral helper construct, and either AAV2mut or, as positive control, the parental wild-type AAV2 helper plasmid ( Fig. 1B ). [5] These findings agree with the reduced AAP2 protein levels observed at late time points of AAV virus production in insect cells ( Fig. 2C ) and with the short AAP2 half-life ( Fig. 3G ), substantiating our conclusion that the viral mechanisms may be similar in both species. [5] The representative Western blots (from 10 ?g of total proteins extracted from cell pellets at each time point) illustrate the rapid decline of AAP2 steady-state levels in the presence of CHX. [5] When we compared AAP2 localization in HEK293T cells transfected with HA-tagged AAP2 or AAP2 C-terminally fused to GFP, using an anti-HA antibody or our polyclonal anti-AAP2 antibody for detection of the HA-AAP2 protein, we observed identical, predominantly nucleolar localization in all cases (data not shown). [5] VP1 to VP3 proteins were detected with the B1 antibody. (C) Quantification of relative GFP expression in HEK293T cells at 48 h after transduction with AAV crude cell lysates (in principle, the same samples as for panel B). [5] Normal cells might be able to dial protein production up or down by adjusting the numbers of these specialized factories, providing “a new layer of control of gene expression,” Barna says. [11] The latter allows for expression of Rep78/52 and VP1/VP2/VP3 proteins in Sf9 cells from the AAV2 rep and cap ORFs, respectively, that were optimized as described by Smith et al. ( 23 ). [5] Although ribosomes are costly, they are essential for translating the genetic code, carried in messenger RNA (mRNA) molecules, into all the proteins the cell needs. “Life evolved around the ribosome,” Dinman says. [11] In their latest study, published online last week in Molecular Cell, Barna and her team determined the abundances of various ribosome proteins with a method known as selected reaction monitoring, which depends on a type of mass spectrometry, a technique for sorting molecules by their weight. [11] A second type churned out all the proteins that allow cells to use vitamin B12, an essential molecule for metabolism. [11] One is Brownian motion — cells are mostly water, and water molecules are always on the move — so inside a cell, proteins and RNA are always jiggling around and jostling. [1] For experimental confirmation, we again transfected HEK293T cells as for Fig. 1B but now arrested protein translation by adding cycloheximide (CHX) 24 h after transfection ( Fig. 3C ). [5] Such tailored ribosomes could provide a cell with another way to control which proteins it generates. [11] Ribosome biogenesis encompasses six important steps (yellow boxes): (i) transcription of components (rRNAs, mRNAs encoding ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs), and snoRNAs); (ii) processing (cleavage of pre-rRNAs); (iii) modification of pre-RNAs, RPs and RBFs; (iv) assembly; (v) transport (nuclear import of RPs and RBFs; pre-ribosome export to the cytoplasm); and (vi) quality control and surveillance. [1] It also remains possible that a key function of AAP is promotion of nucleolar VP transport to facilitate interaction with nucleolar proteins which in turn chaperone AAV capsid assembly. [5] We comprehensively studied AAP-dependent expression of AAV genes and proteins by reverse transcription-PCR, Western blotting, dot blotting, and immunofluorescence, and we quantified capsid assembly by immunostaining with capsid-specific antibodies. [5] While the exact mechanisms have yet to be elucidated, it was proposed that AAP promotes translocation of AAV2 VP3 proteins to the nucleolus, where AAV2 capsid assembly occurs. [5] This has also been implied by the Nakai lab, who interpreted their notion of AAP-independent AAV5 capsid assembly as a potential by-product of the superphysiological VP3 protein expression from their particular construct ( 21 ). [5]

Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. [5] As in all evolution there have evolved exceptions, with helper proteins that checks for erroneous assembly or folding and either unravel or destruct the dysfunctional products. [1] The proteins are synthesized in the cytoplasm and reimported to the nucleus for assembly. [1]

LIVE SCIENCE The protein responsible for a complex type of cell suicide has an accomplice. [4] Scientists pinpoint potential treatment target for pediatric liver cancer NEWS MEDICAL A protein in the cell nucleus already targeted therapeutically for several types of cancer has now been linked to an aggressive form of pediatric liver cancer called hepatoblastoma (HBL), according. [4] Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells. [6] Western blotting was performed from 10 ?g of total proteins extracted from cell pellets of uninfected (c, control) or infected Sf9 cells recovered every 24 h postinfection. [5] It’s now thought there are other things in the cell, that can help proteins etc come together, in the right way. [1] Dry cell pellets were stored at ?20C for protein extraction. [5] Strikingly, heterochromatin protein 1 (HP1) displayed remarkably transient residence times (?10-20 s) in live cells compared with core histones (approximately hours; Cheutin et al., 2003 ), suggesting a rather dynamic maintenance of heterochromatin in live cells. [6] Because of the high packing density of intracellular, and particularly intranuclear, proteins and the lack of tools for sparse labeling, reliable intracellular single-molecule imaging even in fixed cells was not possible until the discovery of photoactivatable or photoswitchable FPs (or dyes; Patterson and Lippincott-Schwartz, 2002 ). [6] More recent evidence suggests that such simple repetitive amino acid motifs, now referred to as low-complexity domains (LCDs), are found to be highly prevalent in a variety of regulatory proteins including many classical TFs and other proteins such as Fused in sarcoma (FUS), TAF15, and Ewing sarcoma protein (EWS; Kwon et al., 2013 ) and can participate in forming dynamic phase-separated compartments in live cells ( Patel et al., 2015 ; Shin and Brangwynne, 2017 ). [6] A photoactivatable GFP for selective photolabeling of proteins and cells. [6] Two-step process underpins upkeep of key protein in cell division SCIENCE DAILY Two complementary processes enable a critical protein, known as CENP-A, to be replenished to enable cells to divide, detailed analysis shows. [4] How Tau aggregates can contribute to cell death in Alzheimer’s disease SCIENCE DAILY New evidence suggests a mechanism by which progressive accumulation of Tau protein in brain cells may lead to Alzheimer’s disease. [4] New mechanism by which Alzheimer’s disease spreads through the brain discovered SCIENCE DAILY The waste-disposal system in a cell can spread harmful protein aggregates between neurons in the brain in Alzheimer’s disease. [4] Waste-disposal system of cell appears to play key role in spread of Alzheimer’s disease NEWS MEDICAL The waste-disposal system in a cell can spread harmful protein aggregates between neurons in the brain in Alzheimer’s disease, according. [4] Two-step process underpins upkeep of key protein in cell division PHYS.ORG Scientists have shed light on a key aspect of healthy cell division, helping build a clearer picture of the complex mechanisms involved. [4] Yeast cells producing a bacterial symmetric protein complex with eight units. [12] Molecular biologist Harry Noller of UC Santa Cruz doubts that cells would evolve to reshuffle the array of proteins in the organelles. [11]

This book provides comprehensive insights into interaction and biosynthesis of proteins, structure of RNA and DNA, polymerase chain reaction, cloning, proteinase activities, and risk factors of hyper-dyslipidemia, etc. The topics included in this book provide a significant overview of the various genetic structures and gene transfer mechanisms. [13] Consistent with this TF hub model, other well-described phase-separated structures known as nuclear bodies (e.g., nuclear speckles, promyelocytic leukemia protein bodies, and Cajal bodies) are thought to be capable of influencing genome organization by sequestering target genes in specialized microenvironments ( Brown et al., 2008 ; Ching et al., 2013 ; Wang et al., 2016a ). [6] Plasmid pMB-eGFP-Puro is derived from the pFastBac plasmid (Thermo Fisher Scientific) and contains a human CMV promoter, the enhanced green fluorescent protein (eGFP) reporter gene, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), a puromycin resistance sequence, and the 3? untranslated region of the human hemoglobin beta (HBB) gene. [5] A mammalian cell may harbor as many as 10 million ribosomes, and it can devote up to 60% of its energy to constructing them from RNA and 80 different types of proteins. [11] Ribosomes, which build a protein (black) from an RNA strand (blue), may specialize in making particular sets of proteins. [11] The ribosome is half protein and half RNA. (Some sources say 2/3 RNA and 1/3 protein but bear with me). [1] “Nomura and his colleagues managed completely to disassemble and then reassemble the 30S ribosomal subunit of E. coli, demonstrating that the 22 parts of the subunit (a 16S RNA molecule and 21 different proteins) contained all the information needed to assemble all the parts into a functional whole (4). [1] Later, the Nomura lab achieved the total reconstitution of functional 50S ribosomal subunits of B. stearothermophilus from a mixture of separated components (two RNA molecules and about 30 different proteins) (5). [1]

Tobacco Mosaic Virus is just one or two types of proteins, plus 1 piece of RNA. You can Separate these and purify them. [1] Viruses were popular, as they were very simple often just 1 RNA strand and 1 type of protein. [1] One type of ribosome built several proteins that control growth, for example. [11] That each ribosome focused on proteins crucial for a certain function took the team by surprise, Barna says. [11]

AAP proteins were detected using an anti-HA antibody binding to an N-terminal HA tag present in all AAP expression constructs. [5] AAP proteins of different AAV serotypes show distinct intracellular localization patterns. [5] Proteins VP1 to VP3 were also expressed from the AAP2-deficient BEV but eventually declined, culminating in almost complete loss of VP-specific signals in the absence of AAP at harvest time (96 h postinfection). [5] After boiling for 5 min at 95C, they were separated on either 8% (VP1, VP2, VP3, and actin) or 12% (AAP) resolving sodium dodecyl sulfate (SDS)-polyacrylamide gels together with a standard protein ladder (PageRuler Plus prestained protein ladder; Thermo Fisher Scientific). [5] In this seminal work, Sonntag et al. identified a previously overlooked protein of 23 to 26 kDa that is encoded in the second open reading frame (ORF) of the AAV2 cap gene and that overlaps with the VP2 and VP3 N termini. [5]

The high stringency of our anti-AAP2 antibody in insect cell lysates allowed us also to study AAP2 stability in the presence of CHX. As exemplified in Fig. 3G, we found that AAP2 undergoes a rapid turnover also when protein synthesis is blocked. [5] Early pioneering studies ( McNally et al., 2000 ; Stenoien et al., 2001 ) to measure TF diffusion and binding dynamics relied on fluorescent proteins (FPs; Tsien, 1998 ) and the use of FRAP assays ( Fig. 1 B ; Axelrod et al., 1976 ). [6]

Curiously, data on non-AAV2 AAP proteins remain very sparse. [5] How ‘gatekeepers’ to a cell’s nucleus let genetic instructions pass through PHYS.ORG Standing guard between a cell’s nucleus and its main chamber, called the cytoplasm, are thousands of behemoth protein structures called nuclear pore complexes, or NPCs. [4] Direct visualization of dynamic structures of protein disaggregation molecular machines PHYS.ORG ClpB, an ATP-fueled protein molecular machine, disentangles and reactivates aggregated proteins. [4]

Structural biology: Until the last cut PHYS.ORG Ribosomes are the cell’s protein factories. [4] The standard view has been that a ribosome doesn’t play favorites with mRNAs–and therefore can synthesize every protein variety. [11] The specialized ribosomes often concentrated on proteins that worked together to perform particular tasks. [11] Molecular and developmental biologist Maria Barna of Stanford University in Palo Alto, California, and colleagues reported in 2011 that mice with too little of one ribosome protein have short tails, sprout extra ribs, and display other anatomical defects. [11] That pattern of abnormalities suggested that the protein shortage had crippled ribosomes specialized for manufacturing proteins key to embryonic development. [11] For one thing, he says, measuring the concentrations of proteins in naturally occurring ribosomes has been difficult. [11] Among 76 ribosome proteins the scientists measured with another mass spectrometry-based method, seven varied enough to indicate ribosome specialization. [11]

I would like to elaborate on the part “Biochemistry works this way, proteins and RNA know how to fold up correctly.” [1] Long story short, it all happens automatically, it’s like putting a thousand piece jigsaw puzzle in a big box, shaking gently for 24 hours, and then opening the box to find the puzzle all completed! Biochemistry works this way, proteins and RNA know how to fold up correctly. [1] The proteins and RNA find each other, and by trial and error, come together and stick to each other, to form the correct shape. [1]

Molecular dissection of human Argonaute proteins by DNA shuffling. [5] This 3-protein complex then “recruits” a molecule of protein 4, etc. Meanwhile proteins 11 and 12 find each other. [1] The team initially created proteins with three mutations to two different sticky amino acids and observed LEGO-like self-assembly in both cases. [12] The ATG codon of VP1 was mutated to ACG in order to produce VP1-VP2-VP3 proteins in insect cells in a stoichiometry close to 1:1:10. [5] For analysis of proteins in insect cells, 10 ?g total protein was extracted in radioimmunoprecipitation assay (RIPA) buffer and loaded onto Novex 10% Tris-glycine polyacrylamide gels (Thermo Fisher Scientific). [5]

Because molecular biology is mostly done in English the proteins of the large subunit are named L-1 to L-31 and the proteins of the small subunit are S-1 to S-21. [1] They only catalyze the polymerization of amino acids into proteins using the mRNA to direct the selection of amino acids. [1] It was reported that dimeric GR has a residence time of ?1.45 s by using a EOS2 fusion protein ( Gebhardt et al., 2013 ), whereas the GR-stable binding residence time measured by using the more photostable HaloTag is several-fold longer (?7.4 s; Swinstead et al., 2016 ). [6] It is possible that these enhancer clusters could serve as multivalent docking sites for dynamic TF recruitment via weak protein-protein interactions potentially directed by LCD-containing proteins. [6] FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know? Curr. [6]

FIG 9 Model for the role of AAP and for its relationship with AAV capsid protein expression and assembly. [5] FIG 6 Proof that AAP fosters assembly of capsids of multiple AAV serotypes. (A) Capsids of the eight AAV serotypes shown were produced by cotransfecting HEK293T cells with the corresponding wild-type or AAP-deficient (mutant) AAV helper, an AAV vector, and an adenoviral helper. [5] We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. [5]

As shown in the original work and solidified in two follow-up studies from the same group ( 18, 19 ), this protein plays a pivotal role in the assembly of AAV2 capsids; hence, it was dubbed assembly-activating protein (AAP). [5] IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. [5]

As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP’s role in and relevance for assembly of genuine AAV capsids have remained largely unclear. [5] Previous work has almost exclusively studied the role of AAP in AAV capsid assembly in the context of virus-like particles that were composed of merely the major capsid protein VP3 ( 17 – 21 ). [5]

Efficient AAV capsid formation may ultimately rely on a combination of VP protein expression above a certain threshold followed by, or concurrent with, AAP-mediated assembly into intact and stable capsids. [5] We believe that this stems from VP stabilization through assembly; hence, the VP proteins detected by Western blotting in the presence of AAP2 were mostly derived from AAV2 particles that were denatured during sample preparation and thus regained reactivity with the B1 antibody. [5] Accordingly, we believe that the main function of AAP2 is not to directly stabilize VP proteins, because this alone would not suffice to foster particle assembly. [5]

FIG 3 Determination of the half-lives of AAV2 VP proteins and of AAP2. (A and B) Quantification of AAV2 VP proteins (A) or assembled particles (B) in microscopy pictures (not shown) of cells stained with the B1 or A20 antibody at 48 h posttransfection. [5] Based on our finding that AAP2 knockout led to a reduction of intracellular VP levels in mammalian cells ( Fig. 1D ), we also analyzed VP protein expression during AAV2 vector production in insect cells in the presence or absence of AAP2. [5] Together with data that steady-state AAP2 levels are influenced by coexpressed capsid proteins ( 19 ), a picture emerges where expression and localization of AAP and VP proteins are tightly coordinated and affect each other in a spatiotemporal yet transient manner ( Fig. 9 ). [5] Despite the simplicity and usefulness of the VP3 model system, drawbacks are that it properly reflects neither (i) endogenous VP protein levels in mammalian cells, due to the strong ectopic promoter used for VP3 expression, nor (ii) the interplay of AAP with the three individual capsid proteins or with genuine capsids composed of VP1 to VP3. [5]

The central aim of the present work was to analyze AAP biology in mammalian and insect cells during production of recombinant AAV vectors based on complete viral capsids comprising all three capsid proteins, VP1 to VP3. [5]

The AAV capsid consists of 60 viral proteins (VPs) which form an 18- to 26-nm icosahedral particle with T1 symmetry ( 3 ) that encapsidates a 4.7-kb-long, single-stranded viral DNA. The three proteins VP1, VP2, and VP3 are represented in the capsid in an approximate ratio of 1:1:10, which is determined by the RNA splicing frequency and by varying translation efficiencies due to alternative start codons within the AAV cap gene. [5] It remained unclear whether AAP is crucial for AAV vector production in mammalian cells at all, as nearly all data published thus far have been obtained with artificial AAV virus-like particles that were composed of only the major capsid protein VP3. [5] While additional studies are needed to identify the exact nature of these bands, we cautiously interpret their specific appearance in the absence of AAP as hints that posttranslational modification, possibly ubiquitination, may be involved in capsid protein destabilization during AAV vector production in insect and mammalian cells. [5] Therefore, Sf9 cell pellets were collected after dual-BEV infection every day, and total protein was extracted and subjected to detection of AAP and VP proteins by Western blotting ( Fig. 2C ). [5] Congruent with the case for AAV2, we found that the other seven tested serotypes (AAV1, AAV3 to -6, and AAV8 and -9) also expressed a steady-state level of VP proteins even in the absence of AAP ( Fig. 7, left columns). [5] It is noteworthy that the MG-132-induced increase in VP protein levels did not result in intact vector particles for any of the serotypes studied here, implying that VP proteins do not spontaneously self-assemble and form infectious particles in cells once a certain steady-state level has been reached. [5] The data at this point showed not only that AAP2 is required for formation of functional AAV2 vector particles in two heterologous systems but that its presence also correlates with the detection of VP protein signals by Western blotting. [5] AAV2 vectors were then produced in spinner flasks after infection of Sf9 insect cells with two BEV: (i) BEV-AAV carrying an enhanced GFP (eGFP) or an alpha-1-antitrypsin (A1AT) transgene flanked by AAV2 ITR sequences and (ii) BEV-Rep2Cap2 expressing AAV2 Rep78/52 and the three VP proteins, with the AAP2 ORF either being intact (AAV2wt) or carrying the knockout mutations (AAV2mut). [5] AAP2 is also crucial for AAV2 vector production and VP protein stability in insect cells. [5] Western blotting was performed, and AAP2 and VP proteins were detected, as for panel C. SDS-polyacrylamide gel loading and BEV infection were verified with anti-GAPDH and anti-GP39 antibodies, respectively. (E) Detection of B1 antibody-reactive, high-molecular-weight bands in mammalian cells expressing AAV2 VP proteins in the absence of AAP2. [5] The finding that AAP2 not only controls AAV2 particle production but also affects levels of capsid proteins in transfected mammalian cells was unexpected. [5]

Surprisingly, Western blot analysis of cell lysates from the samples for Fig. 1C revealed a pronounced reduction in the amounts of all three capsid proteins (VP1, VP2, and VP3) expressed from AAV2mut compared to the wild-type AAV2 control ( Fig. 1D, top gels). [5] The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. [5] AAP is critical for AAV vector production and for VP protein stability. [5] Total VP protein levels were stable in the absence of CHX, regardless of the presence or absence of AAP ( Fig. 3D, two right blots). [5] The AAP-dependent effect was specific for the VP proteins, since levels of Rep proteins remained constant under all conditions ( Fig. 1D, middle gels). [5]

To begin to dissect the mechanisms underlying the loss or increase in VP proteins, we analyzed levels of all major AAV mRNA transcripts. [5] This suggested that AAP2 knockout affects neither transcription nor steady-state levels of cap mRNAs and in turn implied that (de)stabilization occurs on the VP protein level. [5] Even without adding AAP2, VP protein amounts from AAV2mut became identical to those from wild-type AAV2 control after proteasome inhibition (5 and/or 24 h posttransfection). [5] We then asked whether inhibition of proteasomal degradation would affect VP protein localization to the nucleus and the nucleoli, where AAV2 capsid assembly occurs ( 30 ). [5] Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. [5] As reported ( 41, 42 ), the VP1 and VP2 capsid proteins of MVM form cytoplasmic homotrimeric (3VP2) or heterotrimeric (2VP2/1VP1) assembly intermediates which then exploit a structured, ?-stranded nuclear localization motif (NLM) for entry in the nucleus and completion of the assembly process. [5] Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection. [5]

FIG 2 Role of AAP in AAV2 assembly in insect cells. (A) Generation of AAP2 knockout recombinant baculovirus. [5] Until now, the function of AAP during the assembly of recombinant AAV particles in insect cells has not been investigated. [5] ADK/A20 signals were hardly detected for the other five serotypes in the absence of their AAP. We conclude from the sum of our data from the immunofluorescence, dot blot, and transduction analyses that not only AAV2 but at least 10 different natural AAV serotypes strictly require AAP for proper assembly of intact and infectious particles, albeit the extent of this dependency may vary, as implied by the AAV4 and AAV5 data. [5] An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. [5] FIG 7 Further evidence for the varying dependence of AAV serotypes on AAP for assembly of capsid-like structures. [5] The Nakai group investigated the ability of AAP from AAV1 to AAV12 to support assembly of AAV serotypes 1 to 12 ( 21 ). [5]

The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. [6] Depletion of Esrrb and Stat3 had no significant effect on the binding of Sox2 and Oct4 to the enhancer, adding further support for an ordered assembly process at least for some enhancers in ES cells. [6] Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. [6]

A viral assembly factor promotes AAV2 capsid formation in the nucleolus. [5] In the presence of AAP2, we also detected abundant A20 signals, indicating the expected assembly of intact capsids. [5] Note the strong signals with the ADK4 or ADK5 antibodies (detecting assembled AAV4 or AAV5 capsids, respectively), substantiating the evidence for AAP-independent assembly of capsid-like, ADK-reactive structures. [5] Nuclear transport of trimeric assembly intermediates exerts a morphogenetic control on the icosahedral parvovirus capsid. [5]

To corroborate these functional data with analyses of AAP-dependent assembly of physical particles derived from the different serotypes, we performed nondenaturing dot blotting ( Fig. 6A ). [5] It is not even known whether AAP is expressed at all in this heterologous context and whether it is required for correct particle assembly. [5] Altogether, the distinct and often discordant localization patterns of AAP, VP, and capsids that have now been consistently observed in multiple independent studies imply that nucleolar factors are generally not essential for AAV capsid assembly. [5] Further data from Naumer et al. suggest that AAP induces a conformational alteration in VP3, indicating a function as scaffold that nucleates AAV capsid assembly ( 18 ). [5] Deletions of either the hydrophobic AAP N terminus or of the C terminus of VP3 impact AAV2 capsid assembly, implying that these two domains mediate direct AAP-VP interaction. [5]

We noted that cells expressing AAP2 often also stained positive with the A20 antibody (indicative of AAV2 capsid assembly). [5] As a whole, our data demonstrate that in both mammalian and insect cells, (i) AAP2 is expressed from the AAV2 cap gene in similar manners, (ii) AAP2 is strictly required for AAV2 capsid assembly and vector particle production, (iii) AAP2 knockout phenotypes can be restored by AAP2 trans -complementation, and (iv) AAP2 overexpression does not boost AAV2 vector production. [5] Collectively, our results show that AAP2 knockout in the recombinant baculovirus Rep2Cap2 precludes capsid assembly and vector particle production in insect cells, validating and expanding our findings in mammalian cells. [5]

In the same study, AAP2 was also used to rescue capsid assembly from serotypes 1, 2, 5, 8, and 9. [5] This is consistent with our data in Fig. 5B and may imply that AAV3 capsid assembly depends on the presence and the amount of AAP even more critically than that of the other serotypes. [5] Capsid assembly may rapidly occur or at least be triggered at the site of AAP localization, which itself is determined by the AAV serotype. [5] Most AAV serotypes strictly require AAP for capsid assembly. [5]

Arguing against this, at least as an exclusive mechanism, is that assembly of AAV serotypes other than AAV2 can occur outside the nucleolus and that AAP-independent targeting of AAV2 VP3 to the nucleolus via appropriate peptides did not foster AAV2 capsid assembly ( 17, 21 ). [5] Our finding, moreover, is in line with and extends data obtained by Naumer et al. ( 18 ) and Earley et al. ( 21 ), both of whom noted an inability of AAP5 to efficiently rescue assembly of AAV2 virus-like-particles composed of VP3. [5]

The bottom panel is adapted from Fukaya et al. (2016) with permission. (C) Live-cell imaging data suggest that RNA Pol II functions at the core promoter by rapid assembly and disassembly. [6] TFs bound within the assembly mediate distinct functions (e.g., signal transduction and interplay with core promoter), suggesting a functional division of labor for TFs. (B) The TF temporal occupancy pattern at a specific target site is exquisitely regulated by TF concentration in the nucleus and TF residence times at the target site (left). [6]

Hierarchically ordered TF assembly at enhancers enables rapid and flexible regulatory outputs with high specificity ( Fig. 2 ). [6] We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach. [4] Subcellular compartmentalization of adeno-associated virus type 2 assembly. [5] The assembly of multiple TFs at distal enhancers is a signature feature of gene regulation in metazoans. [6] Later, Earley et al. ( 20 ) speculated that AAP5 might lack a nucleolar localization signal due to an overall low net positive charge and that AAV5 assembly might thus occur outside the nucleolus. [5] We simply started with an existing structure and found a simple strategy to induce the assembly of filaments. [12]

It remains possible that VP protein levels are an important determinant of capsid assembly as well. [5] The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes. [5] Adeno-associated virus assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11. [5]

Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein. [5] It is interesting to note that MG-132 treatment had no effect on intracellular VP localization, showing that VP protein stabilization per se is also not sufficient for nuclear or nucleolar AAP trafficking. [5] It uses a nonconventional CTG start codon that is embedded between the weak ACG start codon of VP2 and the strong VP3 ATG, suggesting a temporally coordinated expression pattern of AAP and VP proteins. [5] The AAP ORF fully overlaps that of the VP proteins in all AAV serotypes, implying that diversification of the VP ORF may inadvertently also yield nonnatural AAP proteins. [5] Intriguingly, we have previously reported that a genuine AAV5 helper plasmid, expressing the three VP proteins from the physiological p40 promoter, also results in VP levels much higher than those for various other AAV serotypes ( 31, 37 ). [5] Possibly, in the absence of AAP5, AAV5 VP proteins can self-assemble into aggregates that expose the epitope of the ADK5 antibody (raised against assembled AAV5 capsids). [5] AAP2 or VP proteins were detected using the anti-AAP2 or polyclonal anti-VP antibodies, respectively. [5] Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. [5] We identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. [5] Relevance of assembly-activating protein for adeno-associated virus vector production and capsid protein stability in mammalian and insect cells. [5] Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. [5] Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. [5]

An additional complication is that all prior studies had used artificial AAV capsids that were composed merely of the major capsid protein VP3 expressed from a robust CMV promoter ( 17 – 21 ). [5]

As noted above, the B1 antibody binds VP proteins from AAV serotypes 1 to 3 and 5 to 9 but not AAV4. [5] Intracellular localization of adeno-associated viral proteins expressed in insect cells. [5] To detect viral proteins via Western blotting, 2SDS sample buffer was added to the samples. [5]

Properties of the adeno-associated virus assembly-activating protein. [5]

As expected, A20 staining was negative due to the absence of capsid assembly without AAP2. [5] The mammalian cell cycle regulates parvovirus nuclear capsid assembly. [5]

Therefore, optimization of magnesium is essential for many protein-nucleic acid interactions, ribosome assembly, and the proper function of biological processes of the cell, including protein synthesis. [14] We also noticed that the protein composition of the capsids (42, 37 and 35 kDa) was identical for CaMV and CaMV-TAVm3, indicating that the capsids were properly assembled and the pre-CP properly processed by the viral protease in the cells infected by the mutant virus. [2] Transcription of viral DNA yielded much less 35S and 19S RNAs since the nucleus was probably less re-infected by infectious particles and, consequently, a smaller amount of proteins was synthesized compared to an infection with wild-type CaMV. [2] Analysis of CaMV proteins, DNA, 35S and 19S RNAs, and vsRNAs produced in turnip plants infected with wild-type CaMV or CaMV-TAVm3. [2] Its circular double-stranded DNA genome (~8 kbp) is replicated through reverse transcription of the pre-genomic 35S RNA. The six proteins (P1 to P6) encoded by the CaMV genome are expressed from the polycistronic 35S RNA, whereas P6, also called TAV (TransActivator/Viroplasmin), is primarily synthesized from the monocistronic 19S RNA. TAV (62 kDa) is a nucleocytoplasmic shuttling protein essential for CaMV infectivity. [2]

Having already shown in a previous study that the fusion of EGFP did not modify the capacity of TAV to form EDIBs in BY-2 cells, we observed the cells using LSCM, 16 h and 24 h after transfection. mRFP-TAV protein assembled into large IBs when expressed alone ( Fig 2B, panel 1), thus demonstrating that, as for EGFP, the fusion of mRFP at the N-terminus of TAV did not hinder TAV self-association. [2] When mRFP-TAV was co-expressed with EGFP-TAV in BY-2 cells, the two TAV fusion proteins co-localized, as evidenced by the yellow fluorescent foci observed in the merge image ( Fig 2B, panels 5-7). [2] Further studies using fluorescent microscopy revealed that the P2 protein, the major component of ELIBs, formed small aggregates scattered across the cytoplasm despite being present in high amounts, instead of forming a single large ELIB as in wild-type CaMV-infected cells, suggesting that TAV and/or EDIBs influence the behaviour of P2. [2]

To synthesize proteins of interest, crude extract based CFPS systems harness an ensemble of catalytic components (e.g. RNA polymerases, ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors etc.) that are essential for protein synthesis from cell lysates. [14] Duncan TR, Yahashiri A, Arends SJ, Popham DL, Weiss DS: Identification of SPOR domain amino acids important for septal localization, peptidoglycan binding, and a disulfide bond in the cell division protein FtsN. [15] These viroplasms resemble aggresomes that naturally occur in cells to reduce the toxicity of misfolded proteins and make them susceptible to proteolysis by the proteasome and/or by autophagy. [2] Its complicated cell extract preparation method (periplasmic fraction removal before cell lysis) and relatively low protein yield <100 g/ml leaves room for improvement ( 43 ). [14] For this reason, we hypothesized that increasing cell extract concentration in the CFPS reaction might result in an increase in reporter protein yields, as we previously showed for Streptomyces ( 31 ). [14] Recently, encapsulin nanocompartments have begun to receive considerable interest from bioengineers because of their potential to allow the targeted delivery of drugs, proteins, and mRNAs to specific cells of interest. [16] “Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe”. [16]

We demonstrate that the absence of large EDIBs affects CaMV protein synthesis and capsid assembly, and dramatically reduces CaMV DNA replication efficiency and the subsequent production of infectious particles. [2] TAV, in its aggregated form, might act as a nucleation site for capsid assembly and encapsidation of viral DNA, and in particular as a chaperonin and/or scaffolding protein, since it physically interacts with P4. [2] These results suggest that the EKI mutation in TAV and/or EDIBs has a strong effect on the expression of TAVm3 protein itself but also of P3, and a rather weak or moderate effect on the expression of P1, P2 and P4, and on capsid assembly. [2]

CaMV TAV and TAVm3 proteins expressed in turnip plants and A. thaliana in a viral or non-viral context. [2] The pCK-EGFP vector was used to construct the recombinant plasmids encoding the fusion proteins, consisting of enhanced green fluorescent protein (EGFP) and wild-type or mutant CaMV TAV. Full-length CaMV ORF VI and 3? truncated ORF VI sequences were obtained by PCR performed on pETKaKS.6 recombinant plasmid containing the complete ORF VI of Cabb B-JI isolate with appropriate primers. [2] CaMV TAV, P4, P3, P2 and P1 proteins were detected using polyclonal antisera at a dilution of 1:10,000, alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies (Biosys) at a dilution of 1:5,000 and the colorimetric substrate BCIP (5-Bromo- 4-Chloro-3-Indolyl Phosphate) / NBT (Nitroblue Tetrazolium) detection system (Promega)). [2] CaMV ORF VI sequence was also amplified using two primers carrying at their 5? ends Bsr GI and Xba I sites, respectively, and cloned in these restriction sites of the pmRFP vector, in fusion with the sequence encoding the red fluorescent protein (mRFP). [2] Giband M, Stoeckel ME, Lebeurier G. Use of the immuno-gold technique for in situ localization of cauliflower mosaic virus (CaMV) particles and the major protein of the inclusion bodies. [2] CaMV proteins P1 and P2–P5 could not be tested in the absence of specific antibodies–were also produced at significant levels, indicating that the extremely low amount of TAVm3 was sufficient to efficiently activate reinitiation of 35S RNA translation. [2] The movement protein P1 was expressed at almost similar levels (100% – 88%) in both CaMV- and CaMV-TAVm3-infected turnip plants partly explaining why the systemic propagation of infection occurred at the same rate for CaMV and CaMV-TAVm3. [2] The P2 protein was, on the contrary, produced in relatively high amounts (43%) in CaMV-TAVm3-infected turnip plants ( Fig 7A ), thus indicating that its inability to form ELIBs was due to events independent of its expression level. [2]

Upon demonstration of robust and high yielding protein expression from our P. putida combined transcription and translation system, we then set out to demonstrate the potential for using linear DNA templates for P. putida -based CFPS. High-throughput protein expression has become a key technology in systems biology and synthetic biology. [14] Heterologous proteins ( i. e. interferon) expressed from recombinant CaMV DNA have been detected in proximity of EDIBs. [2] Collagen is a natural protein composed of glycine, alanine, proline, and 16 other kinds of amino acids; its molecular structure is rich in N-H, CO, and -OH and other coordination groups which can be crosslinked with some metal ions. [7] The strong effect of K + on collagen self-assembly belongs to kosmotropes in the Hofmeister series, which can increase the stability of collagen. The binding of K + and Ca 2+ with collagen molecules will lead to the decrease of collagen hydrogen-bonding and the increase of the protein hydrophobicity; and the combination of both ions and collagen leads to a decrease in hydrophobicity on the collagen molecules. [7] Due to electrostatic interaction, numbers of Ca 2+ were adsorbed to the protein, making hydrogen bonds greatly weakened on the polypeptide chains, and finally, the solubility of collagen was decreased. [7]

It has been proposed that EDIBs could be considered as mobile viral factories, which, during the late events of infection, dock and deliver virions to the tubules formed by movement protein P1 in plasmodesmata. [2] Love AJ, Laird J, Holt J, Hamilton AJ, Sadanandom A, Milner JJ. Cauliflower mosaic virus protein P6 is a suppressor of RNA silencing. [2] Laird J, McInally C, Carr C, Doddiah S, Yates G, Chrysanthou E, et al. Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling. [2] Love AJ, Geri C, Laird J, Carr C, Yun BW, Loake GJ, et al. Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity. [2] Angel CA, Lutz L, Yang X, Rodriguez A, Adair A, Zhang Y, et al. The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments. [2] Rodriguez A, Angel CA, Lutz L, Leisner SM, Nelson RS, Schoelz JE. Association of the P6 protein of Cauliflower mosaic virus with plasmodesmata and plasmodesmal proteins. [2] Lutz L, Raikhy G, Leisner SM. Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product. [2] Li Y, Leisner SM. Multiple domains within the Cauliflower mosaic virus gene VI product interact with the full-length protein. [2] Haas M, Geldreich A, Bureau M, Dupuis L, Leh V, Vetter G, et al. The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms. [2]

RANKED SELECTED SOURCES(25 source documents arranged by frequency of occurrence in the above report)

1. (116) Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

2. (86) Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV

3. (20) http://www.quora.com/How-are-ribosomes-and-other-organelles-complex-molecular-machines-formed-I-understand-the-ribosome-can-build-other-complex-nanostructures-like-other-organelles-but-how-is-the-ribosome-itself-made

4. (18) Visualizing transcription factor dynamics in living cells | JCB

5. (16) There are millions of protein factories in every cell. Surprise, they?re not all the same | Science | AAAS

6. (14) Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements | Synthetic Biology | Oxford Academic

7. (13) Observing the cell’s protein factories during self-assembly – PARALLEL STATE

8. (12) SHuffle publications | NEB

9. (11) Cation-Specific Effects on the Self-Assembly of Collagen Molecules Mediated by Acetate on Mica Surface Observed with Atomic Force Microscopy

10. (11) Bio Ch. 19 Flashcards | Quizlet

11. (10) Bio-inspired approach to RNA delivery | MIT News

12. (9) Garcea, Robert L, M.D. | CU Experts | CU Boulder

13. (8) Observing the cell’s protein factories during self-assembly

14. (7) SKKU-BTI Symposium – Soft Matter Research Laboratory

15. (6) Encapsulin nanocompartment – Wikipedia

16. (4) Observing the cell’s protein factories during self-assembly: Electron microscopy images provide important insights into ribosome assembly — ScienceDaily

17. (4) Gideon Dreyfuss | Faculty | About Us | Perelman School of Medicine | Perelman School of Medicine at the University of Pennsylvania

18. (4) The 2017 Nobel Prize in Physiology or Medicine – Press Release

19. (3) LEGO-like proteins revealed

20. (2) A 100-fold leap to GigaDalton DNA nanotech

21. (2) 2018 Intrinsically Disordered Proteins Conference GRC

22. (1) Observing the cell’s protein factories during self-assembly

23. (1) COMPARATIVE PHYSIOCHEMICAL ANALYSIS OF HYDROPHOBINS PRODUCED IN ESCHERICHIA COLI AND PICHIA PASTORIS – ScienceDirect

24. (1) [VIDEO] Our first ever look at bacterial organelle shells – MSU-DOE Plant Research Laboratory

25. (1) A Bacterial Jigsaw Puzzle Is Solved

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