scholarly journals KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin

2019 ◽  
Author(s):  
Hilmar Strickfaden ◽  
Kristal Missiaen ◽  
Michael J. Hendzel ◽  
D. Alan Underhill
Keyword(s):  
Phase Separation ◽  
Spatial Constraint ◽  
Heterochromatin Protein ◽  
Spatial Control ◽  
Conserved Sequence ◽  
Lysine Methyltransferase ◽  
General Phase ◽  
Membrane Bound ◽  
Specific Activities ◽  
Protein Mecp2

AbstractThe pericentromere exists as a distinct chromatin compartment that is thought to form by a process of phase separation. This reflects the ability of the heterochromatin protein CBX5 (aka HP1α) to form liquid condensates that encapsulate pericentromeres.1,2 In general, phase separation compartmentalizes specific activities within the cell, but unlike membrane-bound organelles, their contents rapidly exchange with their surroundings.3 Here, we describe a novel state for the lysine methyltransferase KMT5C where it diffuses within condensates of pericentromeric heterochromatin but undergoes strikingly limited nucleoplasmic exchange, revealing a barrier to exit similar to that of biological membranes. This liquid-like behavior maps to a discrete protein segment with a small number of conserved sequence features and containing separable determinants for localization and retention that cooperate to confer strict spatial control. Accordingly, loss of KMT5C retention led to aberrant spreading of its catalytic product (H4K20me3) throughout the nucleus. We further found that KMT5C retention was reversible in response to chromatin state, which differed markedly for CBX5 and the methyl-CpG binding protein MeCP2, revealing considerable plasticity in the control of these phase separated assemblies. Our results establish that KMT5C represents a precedent in the biological phase separation4 continuum that confers robust spatial constraint of a protein and its catalytic activity without progression to a gel or solid.

scholarly journals The influence of membrane bound proteins on phase separation and coarsening in cell membranes

2012 ◽  
Vol 14 (42) ◽  
pp. 14509 ◽  
Author(s):  
Thomas Witkowski ◽  
Rainer Backofen ◽  
Axel Voigt
Keyword(s):  
Phase Separation ◽  
Cell Membranes ◽  
Membrane Bound

scholarly journals CSIG-27. PRC2-INDEPENDENT FUNCTION OF EZH2/HP1BP3 COMPLEX IN GLIOMA STEM CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi49-vi50
Author(s):  
Junxia Zhang ◽  
Tianfu Yu ◽  
Ning Liu
Keyword(s):  
Stem Cells ◽  
Transcriptional Repression ◽  
Glioma Stem Cells ◽  
Independent Function ◽  
Set Domain ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Histone Lysine Methyltransferase ◽  
Lysine Methyltransferase

Abstract Glioblastoma (GBM) displays cellular and genetical heterogeneity harboring a subpopulation of glioma stem cells (GSCs). Enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, is the core subunit of the polycomb repressor 2 (PRC2) complex, mediates gene transcriptional repression in both normal and tumor stem cells. An oncogenic role of EZH2 as a PRC2-dependent transcriptional silencer is well established; however, non-canonical functions of EZH2 are incompletely understood. Here we found a novel oncogenic mechanism for EZH2 in a PRC2-indenpend way in GSCs. Using HPLC-MS/MS and IP assay, EZH2 bound to HP1BP3 (heterochromatin protein 1 binding protein 3), a heterochromatin-related protein, with its pre-SET domain. Overexpression of H1P3B3 enhanced the proliferation, self-renewal and temozolomide (TMZ) resistance of GBM cells. Intriguingly, H1PBP3 was up-regulated in high grade gliomas with proneural (PN) subtypes and had a high predictive value on prognosis in patients with PN gliomas. Furthermore, EZH2 and HP1BP3 co-activated the expression of WNT7B by blocking the methylation of H3K9, thereby increasing TMZ resistance and tumorigenicity of glioblastoma cells. Interestingly, inhibition of WNT7B autocrine via LGK974, a specific porcupine inhibitor, effectively reversed the TMZ resistance of both GSCs and GBM glioma cells expressing HP1BP3. Hence, targeting the PRC2-independent function of EZH2 is an effective approach to enhance the efficacy of treating GBM.

scholarly journals Assemblages: Functional units formed by cellular phase separation

2014 ◽  
Vol 206 (5) ◽  
pp. 579-588 ◽  
Author(s):  
Jeffrey A. Toretsky ◽  
Peter E. Wright
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Intrinsic Disorder ◽  
Low Complexity ◽  
Disordered Proteins ◽  
Intracellular Space ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disease States ◽  
Membrane Bound

The partitioning of intracellular space beyond membrane-bound organelles can be achieved with collections of proteins that are multivalent or contain low-complexity, intrinsically disordered regions. These proteins can undergo a physical phase change to form functional granules or other entities within the cytoplasm or nucleoplasm that collectively we term “assemblage.” Intrinsically disordered proteins (IDPs) play an important role in forming a subset of cellular assemblages by promoting phase separation. Recent work points to an involvement of assemblages in disease states, indicating that intrinsic disorder and phase transitions should be considered in the development of therapeutics.

scholarly journals Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic ArchaeonPyrococcus furiosus

2001 ◽  
Vol 183 (2) ◽  
pp. 716-724 ◽  
Author(s):  
Michael W. W. Adams ◽  
James F. Holden ◽  
Angeli Lal Menon ◽  
Gerrit J. Schut ◽  
Amy M. Grunden ◽  
...  
Keyword(s):  
Growth Rates ◽  
Elemental Sulfur ◽  
Pyrococcus Furiosus ◽  
Growth Conditions ◽  
Growth Media ◽  
Hyperthermophilic Archaeon ◽  
Order Of Magnitude ◽  
Membrane Bound ◽  
Peptide Metabolism ◽  
Specific Activities

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosusgrows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Nuclear membrane-bound UDPglucuronosyltransferase of rat liver

1982 ◽  
Vol 60 (10) ◽  
pp. 972-979 ◽  
Author(s):  
Jan Zaleski ◽  
Surendra K. Bansal ◽  
Teresa Gessner
Keyword(s):  
Endoplasmic Reticulum ◽  
High Activity ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Glucuronic Acid ◽  
Microsomal Enzyme ◽  
Subcellular Membrane ◽  
Membrane Bound ◽  
Specific Activities

Some properties of rat liver nuclear membrane-bound UDPglucuronosyltransferase were compared with those of the endoplasmic reticulum bound enzyme. The activity of nuclear membrane-bound UDPglucuronosyltransferase was stimulated only about 1.5-fold by Lubrol WX. Under the same conditions microsomal UDPglucuronosyltransferase was, as usual, highly activated (up to 10-fold), when 4-nitrophenol was the acceptor of glucuronic acid. Specific activities of the detergent-activated enzyme were similar in microsomal and nuclear membrane preparations, when the following aglycone substrates were used: 4-methylumbelliferone, 4-nitrophenol, 1-naphthol, phenolphthalein, and testosterone. Apparent Km values for UDP-glucuronic acid ranged between 0.15–0.25 mM for glucuronidation of 4-nitrophenol and 1-naphthol, by either Lubrol WX activated or non-activated, nuclear membrane-bound UDPglucuronosyltransferase. These values were comparable to those found for detergent activated microsomal enzyme. The results show a similarity in behavior of detergent-activated UDPglucuronosyltransferase regardless of subcellular membrane source and, therefore, suggest the association of the same glucuronosyltransferase with nuclear membrane and endoplasmic reticulum. A possible significance of the presence of high activity of this enzyme in nuclear membrane is discussed.

scholarly journals Programmable Sequences of Reflectin Proteins Provides Evidence for Their Evolution Processes, Tunable Intracellular Localization and Interaction with Cytoskeleton

2021 ◽  
Author(s):  
Junyi Song ◽  
Liu Chuanyang ◽  
Baoshan Li ◽  
Liangcheng Liu ◽  
LIng Zeng ◽  
...  
Keyword(s):  
Phase Separation ◽  
Spatial Organization ◽  
Dynamic Performance ◽  
Intracellular Localization ◽  
Evolutionary Relationship ◽  
Biochemical Properties ◽  
Membrane Bound ◽  
Phase Out ◽  
Intracellular Milieu ◽  
Liquid Liquid Phase Separation

Reflectins are membrane-bound proteins located in cephalopods iridocytes, with repeated canonical domains interspersed with cationic linkers. Scientists keep curious about their evolutionary processes, biochemical properties and intracellular functions. Here, by introducing reflectin A1, A2, B1 and C into HEK-293T cells, these proteins were found to phase out from the crowded intracellular milieu, with distinguished localization preferences. Inspired by their programmable block sequences, several truncated reflectin A1 (RfA1) peptides based on repetition of reflectin motifs were designed and transfected into cells. An obvious cyto-/nucleo-plasmic localization preference was once again observed. The dynamic performance of RfA1 derivatives and their analogic behavior between different reflectins suggest a conceivable evolutionary relationship among reflectin proteins. Additionally, a proteomic survey identified biochemical partners which contribute to the phase separation and intracellular localization of RfA1 and its truncations, as well as the close collaboration between RfA1 and the cytoskeleton systems. These findings indicate that liquid-liquid phase separation could be the fundamental mode for reflectins to achieve spatial organization, to cooperate with cytoskeleton during the regulation of reflective coloration. On the other hand, the dynamic behaviors of RfA1 derivatives strongly recommended themselves as programmable molecular tools.

scholarly journals Intracellular wetting mediates contacts between liquid compartments and membrane-bound organelles

2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Halim Kusumaatmaja ◽  
Alexander I. May ◽  
Roland L. Knorr
Keyword(s):  
Phase Separation ◽  
Membrane Interactions ◽  
Cellular Functions ◽  
P Bodies ◽  
Intracellular Degradation ◽  
Wetting Phenomena ◽  
Membrane Bound ◽  
Membrane Contact ◽  
As Stress ◽  
Protein Rich

Protein-rich droplets, such as stress granules, P-bodies, and the nucleolus, perform diverse and specialized cellular functions. Recent evidence has shown the droplets, which are also known as biomolecular condensates or membrane-less compartments, form by phase separation. Many droplets also contact membrane-bound organelles, thereby functioning in development, intracellular degradation, and organization. These underappreciated interactions have major implications for our fundamental understanding of cells. Starting with a brief introduction to wetting phenomena, we summarize recent progress in the emerging field of droplet–membrane contact. We describe the physical mechanism of droplet–membrane interactions, discuss how these interactions remodel droplets and membranes, and introduce "membrane scaffolding" by liquids as a novel reshaping mechanism, thereby demonstrating that droplet–membrane interactions are elastic wetting phenomena. “Membrane-less” and “membrane-bound” condensates likely represent distinct wetting states that together link phase separation with mechanosensitivity and explain key structures observed during embryogenesis, during autophagy, and at synapses. We therefore contend that droplet wetting on membranes provides a robust and intricate means of intracellular organization.

scholarly journals Physical theory of biological noise buffering by multicomponent phase separation

2021 ◽  
Vol 118 (25) ◽  
pp. e2100099118
Author(s):  
Dan Deviri ◽  
Samuel A. Safran
Keyword(s):  
Phase Separation ◽  
Physical Theory ◽  
Expression Noise ◽  
Genes Encoding ◽  
Heterotypic Interactions ◽  
Experimental Approaches ◽  
Membrane Bound ◽  
Noise Buffering ◽  
Liquid Liquid Phase Separation

Maintaining homeostasis is a fundamental characteristic of living systems. In cells, this is contributed to by the assembly of biochemically distinct organelles, many of which are not membrane bound but form by the physical process of liquid–liquid phase separation (LLPS). By analogy with LLPS in binary solutions, cellular LLPS was hypothesized to contribute to homeostasis by facilitating “concentration buffering,” which renders the local protein concentration within the organelle robust to global variations in the average cellular concentration (e.g., due to expression noise). Interestingly, concentration buffering was experimentally measured in vivo in a simple organelle with a single solute, while it was observed not to be obeyed in one with several solutes. Here, we formulate theoretically and solve analytically a physical model of LLPS in a ternary solution of two solutes (ϕ and ψ) that interact both homotypically (ϕ–ϕ attractions) and heterotypically (ϕ–ψ attractions). Our physical theory predicts how the coexisting concentrations in LLPS are related to expression noise and thus, generalizes the concept of concentration buffering to multicomponent systems. This allows us to reconcile the seemingly contradictory experimental observations. Furthermore, we predict that incremental changes of the homotypic and heterotypic interactions among the molecules that undergo LLPS, such as those that are caused by mutations in the genes encoding the proteins, may increase the efficiency of concentration buffering of a given system. Thus, we hypothesize that evolution may optimize concentration buffering as an efficient mechanism to maintain LLPS homeostasis and suggest experimental approaches to test this in different systems.

Steric Pressure between Membrane-Bound Proteins Opposes Lipid Phase Separation

2013 ◽  
Vol 135 (4) ◽  
pp. 1185-1188 ◽  
Author(s):  
Christine S. Scheve ◽  
Paul A. Gonzales ◽  
Noor Momin ◽  
Jeanne C. Stachowiak
Keyword(s):  
Phase Separation ◽  
Lipid Phase ◽  
Membrane Bound ◽  
Lipid Phase Separation

THE INCORPORATION OF 14C-THREONINE AND 14C-GLUCOSAMINE INTO SUBCELLULAR FRACTIONS AND INTO BOVINE SUBMAXILLARY MUCIN BY SLICES OF BOVINE SUBMAXILLARY GLAND

1967 ◽  
Vol 45 (4) ◽  
pp. 507-522 ◽  
Author(s):  
G. Ross Lawford ◽  
Harry Schachter
Keyword(s):  
Time Course ◽  
Submaxillary Gland ◽  
Subcellular Fractionation ◽  
Time Dependent ◽  
Total Soluble Protein ◽  
Residual Fraction ◽  
Membrane Bound ◽  
Specific Activities ◽  
Bovine Submaxillary Mucin ◽  
Additional Support

Bovine submaxillary gland slices were used to investigate the subcellular site of biosynthesis of bovine submaxillary mucin (BSM). Slices were incubated with 14C-threonine and 14C-glucosamine in Krebs medium III at pH 7.4 and 37 °C for varying periods of time up to 180 minutes. Methods of subcellular fractionation based on those developed for liver proved unsatisfactory for use with submaxillary gland, probably because of the presence of large amounts of highly viscous mucin. A method of subcellular fractionation was developed which yielded five fractions: P (total soluble protein) and C (soluble BSM, purified by Cetavlon precipitation) were prepared from the 16,000 × g supernatant; and R (ribosomes), S (deoxycholate-soluble membrane-bound protein), and a residual fraction D were prepared from the 16,000 × g pellet. The incorporation of both tracers into these fractions was time-dependent and was inhibited by preincubation of the slices for 15 minutes with puromycin. The highest specific activities were recorded in the R and S fractions, implicating them as possible sites of glycoprotein biosynthesis. The time course of 14C-threonine incorporation suggested that protein is made on ribosomes, is transferred to the endoplasmic reticulum, and is then ready for storage or secretion. The time course of 14C-glucosamine incorporation suggested that carbohydrate is linked to the protein both while the protein is still associated with the ribosomes and after it has been released from them. The involvement of ribosomes in carbohydrate attachment to polypeptide received additional support from zone centrifugation analysis on sucrose-density gradients of ribosomes labeled with 14C-glucosamine.

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