scholarly journals SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation

2020 ◽  
Author(s):  
Christopher A Smith ◽  
Paul A Humphreys ◽  
Nicola Bates ◽  
Mark A Naven ◽  
Stuart A Cain ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Cell Function ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Type Ii Collagen ◽  
Cartilage Development ◽  
The Matrix ◽  
Col2a1 Expression ◽  
Key Driver

AbstractEpigenetic modification is a key driver of differentiation and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the ACAN –10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating association of ARID5B with SIRT1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, whilst altering ECM composition and suppressing GAG content during cartilage development. These results suggest that SIRT1 activity can be beneficial to cartilage development and matrix protein synthesis but tailored by addition of other positive GAG mediators.

scholarly journals A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 402 ◽  
Author(s):  
Kristen A. Johnson ◽  
Rudramani Pokhrel ◽  
Melissa R. Budicini ◽  
Bernard S. Gerstman ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Membrane Localization ◽  
Virus Like Particles ◽  
Plasma Membrane Localization ◽  
The Matrix ◽  
Dynamics Simulations

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

scholarly journals A Single Amino Acid Deletion in the Matrix Protein of Porcine Reproductive and Respiratory Syndrome Virus Confers Resistance to a Polyclonal Swine Antibody with Broadly Neutralizing Activity

2015 ◽  
Vol 89 (12) ◽  
pp. 6515-6520 ◽  
Author(s):  
Benjamin R. Trible ◽  
Luca N. Popescu ◽  
Nicholas Monday ◽  
Jay G. Calvert ◽  
Raymond R. R. Rowland
Keyword(s):  
Amino Acid ◽  
Matrix Protein ◽  
Infectious Clone ◽  
Virus Neutralization ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Amino Acid Deletion ◽  
Neutralizing Activity ◽  
The Matrix

Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.

An Immunohistochemical Study of Osteopontin in Pigment Gallstone Formation

2010 ◽  
Vol 76 (1) ◽  
pp. 91-95
Author(s):  
Motohiro Imano ◽  
Takao Satou ◽  
Tatsuki Itoh ◽  
Yoshifumi Takeyama ◽  
Atsushi Yasuda ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Matrix Protein ◽  
Bone Matrix ◽  
Gallstone Formation ◽  
Gallbladder Wall ◽  
Gallbladder Epithelium ◽  
Reticular Pattern ◽  
The Matrix ◽  
Pigment Gallstone

Mucin glycoproteins from the gallbladder epithelium are thought to contribute to the matrix or nucleus of gallstones and other biomineralization systems. The involved acidic glycoproteins have been reported in bile and gallstones. In addition, osteopontin (Opn) is a noncollagenous acidic bone matrix glycoprotein that possesses calcium-binding properties. To investigate the role of Opn in pigment gallstone formation, the involvement of Opn in pigment gallstone formation was studied immunohistochemically in the gallbladder wall and in the stones. Staining for Opn was strongly positive in the epithelium of stone-laden gallbladders and in their stones. The stone-laden gallbladders were infiltrated by macrophages, which intensely stained for Opn. Sections of the pigment stones, under low magnification, showed a lamellar pattern of Opn immunolabeling and showed a reticular pattern under high magnification. Our results indicate that Opn, an acidic glycoprotein from the gallbladder epithelium, seems to be involved in lithiasis. Opn from macrophages and/or the epithelium seems to help form the matrix protein.

C-propeptide of type ii procollagen; a protein associated with cartilage mineralization

1986 ◽  
Vol 44 ◽  
pp. 272-273
Author(s):  
E.R. Lee ◽  
A.R. Poole
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Matrix Protein ◽  
Type Ii Collagen ◽  
Collagen Molecule ◽  
Type Ii ◽  
Amino Acid Sequencing ◽  
Procollagen Type ◽  
Matrix Distribution ◽  
The Matrix

A matrix protein has been identified in both calcifying and noncalcifying cartilage during development, and named chondrocalcin. The concentration of this protein is greatly increased in calcifying cartilage and it appears in the matrix when and where mineralization occurs. Amino acid sequencing has recently shown that chondrocalcin is identical to the C-propeptide of type II procollagen. Type II procollagen is a high molecular weight precursor of type II collagen and it is characterized by amino (NH2) and carboxy (C) propeptide extensions. These nonhelical extensions are normally cleaved extracellularly by proteinases to give the collagen molecule. To investigate the synthesis, secretion and matrix distribution of the C-propeptide, particularly during mineralization, this protein has been localized at the EM level with immunogold techniques.

scholarly journals Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.

1994 ◽  
Vol 126 (6) ◽  
pp. 1611-1623 ◽  
Author(s):  
N Amizuka ◽  
H Warshawsky ◽  
J E Henderson ◽  
D Goltzman ◽  
A C Karaplis
Keyword(s):  
Bone Formation ◽  
Embryonic Stem ◽  
Type Ii Collagen ◽  
Epiphyseal Plate ◽  
Epiphyseal Cartilage ◽  
Endochondral Bone Formation ◽  
Type Ii ◽  
Endochondral Bone ◽  
Cartilage Development ◽  
Calcified Cartilage

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.

scholarly journals Intergenotypic Replacement of Lyssavirus Matrix Proteins Demonstrates the Role of Lyssavirus M Proteins in Intracellular Virus Accumulation

2009 ◽  
Vol 84 (4) ◽  
pp. 1816-1827 ◽  
Author(s):  
Stefan Finke ◽  
Harald Granzow ◽  
Jose Hurst ◽  
Reiko Pollin ◽  
Thomas C. Mettenleiter
Keyword(s):  
Endoplasmic Reticulum ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Virus Release ◽  
Host Tropism ◽  
Adaptive Mutations ◽  
M Proteins ◽  
The Matrix ◽  
Virus Genomes

ABSTRACT Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

scholarly journals Role of the Influenza Virus M1 Protein in Nuclear Export of Viral Ribonucleoproteins

2000 ◽  
Vol 74 (4) ◽  
pp. 1781-1786 ◽  
Author(s):  
Matthew Bui ◽  
Elizabeth G. Wills ◽  
Ari Helenius ◽  
Gary R. Whittaker
Keyword(s):  
Influenza Virus ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Semliki Forest Virus ◽  
Kinase Inhibitor ◽  
The Matrix ◽  
Matrix Protein M1 ◽  
Influenza Virus Replication ◽  
Additional Role

ABSTRACT The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117–130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.

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