The role of liquid–liquid phase separation in regulating enzyme activity

Liquid–liquid phase separation (LLPS) is now recognized as a common mechanism underlying regulation of enzyme activity in cells. Insights from studies in cells are complemented by in vitro studies aimed at developing a better understanding of mechanisms underlying such control. These mechanisms are often based on the influence of LLPS on the physicochemical properties of the enzyme's environment. Biochemical mechanisms underlying such regulation include the potential for concentrating reactants together, tuning reaction rates, and controlling competing metabolic pathways. LLPS is thus a powerful tool with extensive utilities at the cell's disposal, e.g. for consolidating cell survival under stress or rerouting metabolic pathways in response to the energy state of the cell. Here, we examin the evidence for how LLPS affects enzyme catalysis and begin to understand emerging concepts and expand our understanding of enzyme catalysis in living cells.     

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.     

Biophysics of endocytic vesicle formation: A focus on liquid–liquid phase separation

Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid–liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.     

Regulation of synaptic nanodomain by liquid–liquid phase separation: A novel mechanism of synaptic plasticity

Advances in microscopy techniques have revealed the details of synaptic nanodomains as defined by the segregation of specific molecules on or beneath both presynaptic and postsynaptic membranes. However, it is yet to be clarified how such segregation is accomplished without demarcating membrane and how nanodomains respond to the neuronal activity. It was recently discovered that proteins at the synapse undergo liquid–liquid phase separation (LLPS), which not only contributes to the accumulation of synaptic proteins but also to further segregating the proteins into subdomains by forming phase-in-phase structures. More specifically, CaMKII, a postsynaptic multifunctional kinase that serves as a signaling molecule, acts as a synaptic cross-linker which segregates certain molecules through LLPS in a manner triggered by Ca²⁺. Nanodomain formation contributes to the establishment of trans-synaptic nanocolumns, which may be involved in the optimization of spatial arrangement of the transmitter release site and receptor, thereby serving as a new mechanism of synaptic plasticity.     

The role of prostaglandins in regulating vascular smooth muscle cell sur-vival par

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a major causative factor in atherosclerosis. Prostaglandins, secreted by endothelial cells, are reported to attenuate VSMC proliferation, but the mechanism through which this response is mediated is poorly denned. Here, the effect of prostaglandin receptor-selective agonists on the activity status of ERK and PKC, both known to modulate proliferative responses, was determined. The effect of the prostacyclin mimetic, iloprost, at inducing apoptosis was also investigated. VSMCs in culture were shown to express proteins that were detected by antibodies selective     

The role of stratifin in fibroblast–keratinocyte interaction

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a “stemness condition” of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.     

Vapour–liquid phase equilibria of simple fluids confined in patterned slit pores

We present the influence of surface heterogeneity on the vapour–liquid phase behaviour of square-well fluids in slit pores using grand-canonical transition-matrix Monte Carlo simulations along with the histogram-reweighting method. Properties such as phase coexistence envelopes, critical properties and local density profiles of the confined SW fluid are reported for chemically and physically patterned slit surfaces. It is observed that in the chemically patterned pores, fluid–fluid and surface attraction parameters along with the width of attractive and inert stripes play fundamentally different roles in the phase coexistence and critical properties. On the other hand, pillar gap and height significantly affect the vapour–liquid equilibria in the physically patterned slit pores. We also present the effect of chemically and physically patterned slit surfaces on the spreading pressure.     

The role of tumor-specific Lyt-1+2− T cells in eradicating tumor cells in vivo

Summary The present study investigates some of mechanisms for tumor-specific Lyt-1⁺2^– T cell-mediated tumor cell eradication in vivo through analyses of tumor specificity in the afferent tumor recognition and efferent rejection phases. When C3H/He mice which had acquired immunity against syngeneic MH134 hepatoma were challenged with other syngeneic X5563 plasmacytoma cells, these mice failed to exhibit any inhibitory effect on the growth of X5563 tumor cells. However, the inoculation of X5563 tumor cells into the MH134-immune C3H/He mice together with the MH134 tumor cells resulted in appreciable growth inhibition of antigenically distinct (bystander) X5563 tumor cells. Although the growth of X5563 cells was inhibited in an antigen-nonspecific way in mice immunized to antigenically unrelated tumor cells (bystander effect), the activation of Lyt-1⁺2^– T cells leading to this effect was strictly antigen-specific. Such a bystander growth inhibition also required the admixed inoculation of the bystander (X5563) and specific target (MH134) tumor cells into a single site in mice immunized against the relevant MH134 tumor cells. Furthermore, the results demonstrated that Lyt-1⁺2^– T cells specific to MH134 tumor cells were responsible for mediating the growth inhibition of antigenically irrelevant (bystander) and relevant tumor cells. These results are discussed in the context of cellular and molecular mechanisms involved in the Lyt-1⁺2^– T cell-initiated bystander phenomenon.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.