scholarly journals Cryo-EM structure of the Smc5/6 holo-complex

2021 ◽  
Author(s):  
Stephen T Hallett ◽  
Isabella Campbell Harry ◽  
Pascale Schellenberger ◽  
Lihong Zhou ◽  
Nora B Cronin ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Cellular Response ◽  
Essential Role ◽  
Insect Cells ◽  
Budding Yeast ◽  
Viral Integration ◽  
Functional Equivalence ◽  
Restriction Factor ◽  
Protein Core ◽  
Accessory Subunits

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral integration. Here, we report the cryo-EM structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells --providing a full overview of the complex in its apo / non-liganded form, and revealing how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the "W-loop" of Smc4 or "F-loop" of Smc1, mediates an essential interaction with Nse1. Taken together, these data confirm a degree of functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

scholarly journals Protein kinase C group B members PKC-δ, -ɛ, -ζ and PKC-L(η). Comparison of properties of recombinant proteins in vitro and in vivo

1992 ◽  
Vol 283 (3) ◽  
pp. 781-787 ◽  
Author(s):  
M Liyanage ◽  
D Frith ◽  
E Livneh ◽  
S Stabel
Keyword(s):  
Protein Kinase C ◽  
Recombinant Proteins ◽  
Phorbol Ester ◽  
Insect Cells ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Group A ◽  
Group B

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.

scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

scholarly journals Enhanced expression of recombinant proteins in insect cells using a baculovirus vector containing a bacterial leader sequence

1992 ◽  
Vol 20 (22) ◽  
pp. 6111-6112 ◽  
Author(s):  
Timothy C. Peakman ◽  
Ian GiCharles ◽  
Mark A. Sydenham ◽  
Dirk R. Gewert ◽  
Martin J. Page ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Insect Cells ◽  
Leader Sequence ◽  
Baculovirus Vector ◽  
Enhanced Expression

Complex N-glycosylation of Recombinant Proteins by Insect Cells

2002 ◽  
Vol 1 (2) ◽  
pp. 70-73 ◽  
Author(s):  
Laura Palomares ◽  
Octavio Ramírez
Keyword(s):  
Recombinant Proteins ◽  
Insect Cells

Expression and Immunoreactivities of Hepatitis E Virus Genotype 3 Open Reading Frame-2 (ORF-2) Recombinant Proteins Expressed in Insect Cells

2009 ◽  
Vol 1 (2) ◽  
pp. 77-84 ◽  
Author(s):  
Nereida Jiménez de Oya ◽  
Inmaculada Galindo ◽  
Estela Escribano-Romero ◽  
Ana-Belén Blázquez ◽  
Julio Alonso-Padilla ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Hepatitis E Virus ◽  
Insect Cells ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Genotype 3 ◽  
Virus Genotype ◽  
Reading Frame ◽  
Open Reading Frame 2

scholarly journals Cdc48 regulates intranuclear quality control sequestration of the Hsh155 splicing factor in budding yeast

2020 ◽  
Vol 133 (23) ◽  
pp. jcs252551 ◽  
Author(s):  
Veena Mathew ◽  
Arun Kumar ◽  
Yangyang K. Jiang ◽  
Kyra West ◽  
Annie S. Tam ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Essential Role ◽  
Protein Quality ◽  
Budding Yeast ◽  
Protein Quality Control ◽  
Genotoxic Stress ◽  
Splicing Factor ◽  
Dna Complexes ◽  
Partner Proteins

ABSTRACTCdc48 (known as VCP in mammals) is a highly conserved ATPase chaperone that plays an essential role in the assembly and disassembly of protein–DNA complexes and in degradation of misfolded proteins. We find that in Saccharomyces cerevisiae budding yeast, Cdc48 accumulates during cellular stress at intranuclear protein quality control sites (INQ). We show that Cdc48 function is required to suppress INQ formation under non-stress conditions and to promote recovery following genotoxic stress. Cdc48 physically associates with the INQ substrate and splicing factor Hsh155, and regulates its assembly with partner proteins. Accordingly, cdc48 mutants have defects in splicing and show spontaneous distribution of Hsh155 to INQ aggregates, where it is stabilized. Overall, this study shows that Cdc48 regulates deposition of proteins at INQ and suggests a previously unknown role for Cdc48 in the regulation or stabilization of splicing subcomplexes.This article has an associated First Person interview with the first author of the paper.

scholarly journals Anoxia-Induced Suspended Animation in Budding Yeast as an Experimental Paradigm for Studying Oxygen-Regulated Gene Expression

2008 ◽  
Vol 7 (10) ◽  
pp. 1795-1808 ◽  
Author(s):  
Kin Chan ◽  
Mark B. Roth
Keyword(s):  
Signaling Pathway ◽  
Cellular Response ◽  
Prolonged Exposure ◽  
Budding Yeast ◽  
Transcriptional Profiling ◽  
Retrograde Signaling ◽  
Pcr Analysis ◽  
Suspended Animation ◽  
Mitochondrial Retrograde Signaling ◽  
Regulated Gene Expression

ABSTRACT A lack of oxygen can force many organisms to enter into recoverable hypometabolic states. To better understand how organisms cope with oxygen deprivation, our laboratory previously had shown that when challenged with anoxia, both the nematode Caenorhabditis elegans and embryos of the zebrafish Danio rerio enter into suspended animation, in which all life processes that can be observed by light microscopy reversibly halt pending the restoration of oxygen (P. A. Padilla and M. B. Roth, Proc. Natl. Acad. Sci. USA 98:7331-7335, 2001, and P. A. Padilla, T. G. Nystul, R. A. Zager, A. C. Johnson, and M. B. Roth, Mol. Biol. Cell 13:1473-1483, 2002). Here, we show that both sporulating and vegetative cells of the budding yeast Saccharomyces cerevisiae also enter into a similar state of suspended animation when made anoxic on a nonfermentable carbon source. Transcriptional profiling using cDNA microarrays and follow-on quantitative real-time PCR analysis revealed a relative derepression of aerobic metabolism genes in carbon monoxide (CO)-induced anoxia when compared to nitrogen (N2) gas-induced anoxia, which is consistent with the known oxygen-mimetic effects of CO. We also found that mutants deleted for components of the mitochondrial retrograde signaling pathway can tolerate prolonged exposure to CO but not to N2. We conclude that the cellular response to anoxia is dependent on whether the anoxic gas is an oxygen mimetic and that the mitochondrial retrograde signaling pathway is functionally important for mediating this response.

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