TAT-RasGAP317-326 kills cells by targeting inner-leaflet–enriched phospholipids

TAT-RasGAP317–326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317–326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317–326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317–326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317–326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

Review of PIP2 in Cellular Signaling, Functions and Diseases

Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and cell–matrix adhesion. Central to this process is phosphatidylinositol bisphosphate (PIP2). The levels of PIP2 in the membrane are rapidly altered by the activity of phosphoinositide-directed kinases and phosphatases, and it binds to dozens of different intracellular proteins. Despite the vast literature dedicated to understanding the regulation of PIP2 in cells over past 30 years, much remains to be learned about its cellular functions. In this review, we focus on past and recent exciting results on different molecular mechanisms that regulate cellular functions by binding of specific proteins to PIP2 or by stabilizing phosphoinositide pools in different cellular compartments. Moreover, this review summarizes recent findings that implicate dysregulation of PIP2 in many diseases

A Splicing Variant in OCRL Gene Might Explain the Second Case of Lowe Syndrome in Iran

: Lowe syndrome is a condition that primarily affects eyes, brain, and kidneys. This disorder follows X-linked recessive mode of inheritance and it occurs in males mainly. Mutations in OCRL (located at Xq25) gene can cause accumulation of phosphatidylinositol bisphosphate and disturbed actin cytoskeleton remodeling. There are 268 mutations in OCRL gene causing Lowe syndrome or Dent disease 2 in HGMD database, however 10 - 20% of Lowe syndrome suspects remain undiagnosed at molecular level. Here we present a male case of Lowe syndrome with characteristic features. Comprehensive clinical examination and genetic counseling were performed. Sanger sequencing was employed to investigate the possible OCRL mutations and we identified a donor splice site variant (NM-000276: c.2469 + 1G > A) in hemizygous state. This is a pathogenic variant according to the ACMG standards and guidelines and might explain the clinical features of the patient. This result is in accordance with the clinical diagnosis of Lowe syndrome and it is absent from ExAC, 1000 G, Iranome, GME, gnomAD Genome databases of healthy controls. In-silico analysis of this splicing variant revealed that the position is highly conserved between species. Splicing prediction tools predicted some changes in splicing pattern of the OCRL transcript, elimination of some protein features, and malfunctioning the OCRL protein as a consequence of this variant. Accordingly, we proposed the c.2469 + 1G > A variant might explain the clinical features in studied patient and be employed for prenatal diagnosis of Lowe syndrome in the family.

Activation microswitches in GPCRs function as rheostats in cell membrane

Although multiple components of the cell membrane modulate the stability and activation of G protein coupled receptors (GPCRs), the activation mechanism comes from detergent studies, since it is challenging to study activation in multi-component lipid bilayer. Using the multi-scale molecular dynamics simulations(50μs), our comparative study between cell membrane and detergents shows that: the changes in inter-residue distances, known as activation microswitches, show an ensemble of states in the extent of activation in cell membrane. We forward a rheostat model of GPCR activation rather than a binary switch model. Phosphatidylinositol bisphosphate (PIP2) and calcium ions, through a tug of war, maintain a balance between the GPCR stability and activity in cell membrane. Due to the lack of receptor stiffening effects by PIP2, detergents promote more transitions among conformational states than cell membrane. These findings connect the chemistry of cell membrane lipids to receptor activity useful to design detergents mimicking cell membrane.

A melanosomal two-pore sodium channel regulates pigmentation

Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane.