Myo-inositol belongs to one of the sugar alcohol groups known as cyclitols. Phosphatidylinositols are one of the derivatives of Myo-inositol, and constitute important mediators in many intracellular processes such as cell growth, cell differentiation, receptor recycling, cytoskeletal organization, and membrane fusion. They also have even more functions that are essential for cell survival. Mutations in genes encoding phosphatidylinositols and their derivatives can lead to many disorders. This review aims to perform an in-depth analysis of these connections. Many authors emphasize the significant influence of phosphatidylinositols and phosphatidylinositols\' phosphates in the pathogenesis of myotubular myopathies, neurodegenerative disorders, carcinogenesis, and other less frequently observed diseases. In our review, we have focused on three of the most often mentioned groups of disorders. Inositols are the topic of many studies, and yet, there are no clear results of successful clinical trials. Analysis of the available literature gives promising results and shows that further research is still needed.

Cells utilize protein translocation to specific compartments for spatial and temporal regulation of protein activity, in particular in the context of signaling processes. Protein recognition and binding to various subcellular membranes is mediated by a network of phosphatidylinositol phosphate (PIP) species bearing one or multiple phosphate moieties on the polar inositol head. Here, we report a new, highly efficient method for optical control of protein localization through the site-specific incorporation of a photocaged amino acid for steric and electrostatic disruption of inositol phosphate recognition and binding. We demonstrate general applicability of the approach by photocaging two unrelated proteins, sorting nexin 3 (SNX3) and the pleckstrin homology (PH) domain of phospholipase C delta 1 (PLCδ1), with two distinct PIP binding domains and distinct subcellular localizations. We have established the applicability of this methodology through its application to Son of Sevenless 2 (SOS2), a signaling protein involved in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Upon fusing the photocaged plasma membrane-targeted construct PH-enhanced green fluorescent protein (EGFP), to the catalytic domain of SOS2, we demonstrated light-induced membrane localization of the construct resulting in fast and extensive activation of the ERK signaling pathway in NIH 3T3 cells. This approach can be readily extended to other proteins, with minimal protein engineering, and provides a method for acute optical control of protein translocation with rapid and complete activation.

The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.

Phosphatidylinositol phosphates (PIPs) are involved in many cellular events as important secondary messengers. In Entamoeba histolytica, a human intestinal protozoan parasite, virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis are regulated by PIPs. It has been well established that PI3P, PI4P, and PI(3,4,5)P3 play specific roles during amoebic trogo- and phagocytosis. In the present study, we demonstrated the nuclear localization of PI4P in E. histolytica trophozoites in steady state with immunofluorescence imaging and immunoelectron microscopy, using anti-PI4P antibodies and PI4P biosensors [substrate of the Icm/ Dot type IV secretion system (SidM)]. We further showed that the nuclear PI4P decreased after a co-culture with human erythrocytes or Chinese hamster ovary (CHO) cells. However, concomitant changes in the localization and the amount of PI(4,5)P2, which is the expected major metabolized (phosphorylated) product of PI4P, were not observed. This phenomenon was specifically caused by whole or ghost erythrocytes and CHO cells, but not artificial beads. The amount of PIP2 and PIP, biochemically estimated by [32P]-phosphate metabolic labeling and thin layer chromatography, was decreased upon erythrocyte adherence. Altogether, our data indicate for the first time in eukaryotes that erythrocyte attachment leads to the metabolism of nuclear PIPs, and metabolites other than PI(4,5)P2 may be involved in the regulation of downstream cellular events such as cytoskeleton rearrangement or transcriptional regulation.

Luminal pH and the distinctive distribution of phosphatidylinositol phosphate (PIP) lipids are central identifying features of organelles in all eukaryotic cells that are also critical for organelle function. V-ATPases are conserved proton pumps that populate and acidify multiple organelles of the secretory and the endocytic pathway. Complete loss of V-ATPase activity causes embryonic lethality in higher animals and conditional lethality in yeast, while partial loss of V-ATPase function is associated with multiple disease states. On the other hand, many cancer cells increase their virulence by upregulating V-ATPase expression and activity. The pH of individual organelles is tightly controlled and essential for function, but the mechanisms for compartment-specific pH regulation are not completely understood. There is substantial evidence indicating that the PIP content of membranes influences organelle pH. We present recent evidence that PIPs interact directly with subunit isoforms of the V-ATPase to dictate localization of V-ATPase subpopulations and participate in their regulation. In yeast cells, which have only one set of organelle-specific V-ATPase subunit isoforms, the Golgi-enriched lipid PI(4)P binds to the cytosolic domain of the Golgi-enriched a-subunit isoform Stv1, and loss of PI(4)P binding results in mislocalization of Stv1-containing V-ATPases from the Golgi to the vacuole/lysosome. In contrast, levels of the vacuole/lysosome-enriched signaling lipid PI(3,5)P2 affect assembly and activity of V-ATPases containing the Vph1 a-subunit isoform. Mutations in the Vph1 isoform that disrupt the lipid interaction increase sensitivity to stress. These studies have decoded \"zip codes\" for PIP lipids in the cytosolic N-terminal domain of the a-subunit isoforms of the yeast V-ATPase, and similar interactions between PIP lipids and the V-ATPase subunit isoforms are emerging in higher eukaryotes. In addition to direct effects on the V-ATPase, PIP lipids are also likely to affect organelle pH indirectly, through interactions with other membrane transporters. We discuss direct and indirect effects of PIP lipids on organelle pH, and the functional consequences of the interplay between PIP lipid content and organelle pH.

Phosphoinositides are known to play multiple roles in eukaryotic cells. Although dysregulation of phosphoinositide metabolism in the retina has been reported to cause visual dysfunction in animal models and human patients, our understanding of the phosphoinositide composition of the retina is limited. Here, we report a characterization of the phosphoinositide profile of the mouse retina and an analysis of the subcellular localization of major phosphorylated phosphoinositide forms in light-sensitive photoreceptor neurons. Using chromatography of deacylated phosphatidylinositol headgroups, we established PI(4,5)P2 and PI(4)P as two major phosphorylated phosphoinositides in the retina. Using high-resolution mass spectrometry, we revealed 18:0/20:4 and 16:0/20:4 as major fatty-acyl chains of retinal phosphoinositides. Finally, analysis of fluorescent phosphoinositide sensors in rod photoreceptors demonstrated distinct subcellular distribution patterns of major phosphoinositides. The PI(4,5)P2 reporter was enriched in the inner segments and synapses, but was barely detected in the light-sensitive outer segments. The PI(4)P reporter was mostly found in the outer and inner segments and the areas around nuclei, but to a lesser degree in the synaptic region. These findings provide support for future mechanistic studies defining the biological significance of major mono- (PI(4)P) and bisphosphate (PI(4,5)P2) phosphatidylinositols in photoreceptor biology and retinal health.

Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.

Phosphatidylinositol phosphates (PIPs) are lipid signaling molecules that play key roles in many cellular processes. PIP5K1A kinase catalyzes phosphorylation of PI4P to form PIP2, which in turn interacts with membrane and membrane-associated proteins. We explore the mechanism of membrane binding by the PIP5K1A kinase using a multiscale molecular dynamics approach. Coarse-grained simulations show binding of monomeric PIP5K1A to a model cell membrane containing PI4P. PIP5K1A did not bind to zwitterionic or anionic membranes lacking PIP molecules. Initial encounter of kinase and bilayer was followed by reorientation to enable productive binding to the PI4P-containing membrane. The simulations suggest that unstructured regions may be important for the preferred orientation for membrane binding. Atomistic simulations indicated that the dimeric kinase could not bind to the membrane via both active sites at the same time, suggesting a conformational change in the protein and/or bilayer distortion may be needed for dual-site binding to occur.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule-organizing center in a \"cloud,\" and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biological functions, such as cargo degradation, antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biological events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid-protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the molecular machineries that control this mobility.