scholarly journals Bridging-induced phase separation induced by cohesin SMC protein complexes

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5905
Author(s):  
Je-Kyung Ryu ◽  
Céline Bouchoux ◽  
Hon Wing Liu ◽  
Eugene Kim ◽  
Masashi Minamino ◽  
...  
Keyword(s):  
Phase Separation ◽  
Genome Organization ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Yeast Cells ◽  
Base Pairs ◽  
Fundamental Building Block ◽  
Structural Maintenance Of Chromosome

Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

scholarly journals Phase separation induced by cohesin SMC protein complexes

2020 ◽  
Author(s):  
Je-Kyung Ryu ◽  
Celine Bouchoux ◽  
Hon Wing Liu ◽  
Eugene Kim ◽  
Masashi Minamino ◽  
...  
Keyword(s):  
Phase Separation ◽  
Genome Organization ◽  
Weak Interactions ◽  
Protein Complexes ◽  
Yeast Cells ◽  
Liquid Droplets ◽  
Independent Manner ◽  
Reversible Dissociation

AbstractCohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization.One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

scholarly journals FORMULATION AND IN VITRO, IN VIVO EVALUATION OF PRONIOSOMAL GEL OF NEOMYCIN SULPHATE

2019 ◽  
pp. 156-163
Author(s):  
AMOL SHETE ◽  
PRIYANKA THORAT ◽  
RAJENDRA DOIJAD ◽  
SACHIN SAJANE
Keyword(s):  
Phase Separation ◽  
Skin Irritation ◽  
Entrapment Efficiency ◽  
Separation Method ◽  
Gelling Agent ◽  
In Vivo Evaluation ◽  
Skin Delivery ◽  
Span 60

Objective: The objectives of present investigation were to prepare and evaluate proniosomes of neomycin sulphate (NS) by coacervation phase separation method by using sorbitan monostearate (span 60) and lecithin as a surfactant to increase the penetration through the skin and study the effect of concentration of the same. Methods: Proniosomes of neomycin sulphate (NS) were prepared by coacervation phase separation method by using span 60 and lecithin. The effect of concentration of span 60 and lecithin was studied by factorial design. The prepared proniosomes were converted to gel by using carbopol as a gelling agent. The prepared formulations were evaluated for entrapment efficiency, in vitro drug diffusion, in vitro antibacterial activity and in vivo skin irritation test etc. Results: All Formulation showed the percentage entrapment efficiency in the range 38.31±0.05% to 77.96±0.06%, good homogeneity and gel was easily spreadable with minimal of shear. Optimized formulation showed enhanced rate of diffusion in vitro, increase in zone of inhibition against staphylococcus aureus, no skin irritation and showed good stability. Conclusion: The results of present study indicates that proniosomal gel formulated by using combination of span 60, Lecithin, cholesterol can be used to enhance skin delivery of NS because of excellent permeation of drug. Developed proniosomal gel formulation was promising carrier for NS

scholarly journals Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 915
Author(s):  
Jazelli Mueterthies ◽  
Davit A. Potoyan
Keyword(s):  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Bodies ◽  
Disordered Proteins ◽  
Solvent Exposure ◽  
Ion Release ◽  
Dimer Formation ◽  
Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

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