Development of a versatile HPLC-based method to evaluate the activation status of small GTPases

Protein-Protein Interactions (PPIs) drive major signalling cascades and play critical role in cell proliferation, apoptosis, angiogenesis and trafficking. Deregulated PPIs are implicated in multiple malignancies and represent the critical targets for treating cancer. Herein, we discuss the key protein-protein interacting domains implicated in cancer notably PDZ, SH2, SH3, LIM, PTB, SAM and PH. These domains are present in numerous enzymes/kinases, growth factors, transcription factors, adaptor proteins, receptors and scaffolding proteins and thus represent essential sites for targeting cancer. This review explores the candidature of various proteins involved in cellular trafficking (small GTPases, molecular motors, matrix-degrading enzymes, integrin), transcription (p53, cMyc), signalling (membrane receptor proteins), angiogenesis (VEGFs) and apoptosis (BCL-2family), which could possibly serve as targets for developing effective anti-cancer regimen. Interactions between Ras/Raf; X-linked inhibitor of apoptosis protein (XIAP)/second mitochondria-derived activator of caspases (Smac/DIABLO); Frizzled (FRZ)/Dishevelled (DVL) protein; beta-catenin/T Cell Factor (TCF) have also been studied as prospective anticancer targets. Efficacy of diverse molecules/ drugs targeting such PPIs although evaluated in various animal models/cell lines, there is an essential need for human-based clinical trials. Therapeutic strategies like the use of biologicals, high throughput screening (HTS) and fragment-based technology could play an imperative role in designing cancer therapeutics. Moreover, bioinformatic/computational strategies based on genome sequence, protein sequence/structure and domain data could serve as competent tools for predicting PPIs. Exploring hot spots in proteomic networks represents another approach for developing targetspecific therapeutics. Overall, this review lays emphasis on a productive amalgamation of proteomics, genomics, biochemistry, and molecular dynamics for successful treatment of cancer.

Download Full-text

Stochastic activation and bistability in a Rab GTPase regulatory network

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921027117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6540-6549

Author(s):

Urban Bezeljak ◽

Hrushikesh Loya ◽

Beata Kaczmarek ◽

Timothy E. Saunders ◽

Martin Loose

Keyword(s):

Regulatory Network ◽

Regulatory Networks ◽

Membrane Surface ◽

Activity Patterns ◽

Small Gtpases ◽

Signaling Networks ◽

Rab Gtpase ◽

Endomembrane System ◽

In Vitro Reconstitution

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

Download Full-text

TBC proteins: GAPs for mammalian small GTPase Rab?

Bioscience Reports ◽

10.1042/bsr20100112 ◽

2011 ◽

Vol 31 (3) ◽

pp. 159-168 ◽

Cited By ~ 115

Author(s):

Mitsunori Fukuda

Keyword(s):

Insulin Resistance ◽

Small Gtpases ◽

Structure And Function ◽

Small Gtpase ◽

Cell Cycle Regulators ◽

Gtpase Activating Protein ◽

Domain Family ◽

Protein Motif ◽

Conserved Domain ◽

And Function

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.

Download Full-text

Revising Endosomal Trafficking under Insulin Receptor Activation

International Journal of Molecular Sciences ◽

10.3390/ijms22136978 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6978

Author(s):

Maria J. Iraburu ◽

Tommy Garner ◽

Cristina Montiel-Duarte

Keyword(s):

Plasma Membrane ◽

Tyrosine Kinase ◽

Insulin Receptor ◽

Molecular Mechanisms ◽

Receptor Activation ◽

Small Gtpases ◽

Early Endosome ◽

Tyrosine Kinase Receptors ◽

Endosomal Trafficking ◽

Cell Functions

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

Download Full-text

Interactions of Lipid Droplets with the Intracellular Transport Machinery

International Journal of Molecular Sciences ◽

10.3390/ijms22052776 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2776

Author(s):

Selma Yilmaz Dejgaard ◽

John F. Presley

Keyword(s):

Membrane Trafficking ◽

Lipid Droplets ◽

Intracellular Transport ◽

Neutral Lipids ◽

Small Gtpases ◽

Lipid Phase ◽

Intracellular Organelles ◽

And Function ◽

Lipid Phase Separation ◽

Endocytic Pathways

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

Download Full-text