Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways

Target of rapamycin is a Ser/Thr kinase that operates in two conserved multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin, and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and loss of endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics, revealed that TORC2 regulates these readouts via distinct pathways as follows: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2, whereas the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomic screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB_M) can compete with wild-type PLB (PLB_W) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB_W, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K_Ca, measured from the Ca²⁺-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB_W, while the potency of each of the LOF PLB_M was about one third that of PLB_W. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB_W and LOF PLB_M, inhibitory potency was reduced relative to that of PLB_W alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB_W was decreased by both (unlabeled) LOF PLB_M. These results show that LOF PLB_M can compete both physically and functionally with PLB_W, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB_M targeted for gene therapy of heart failure in humans.     

Spotting the right location— imaging approaches to resolve the intracellular localization of invasive pathogens

Background

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.

Scope of Review

Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.

Major Conclusions

We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.

General Significance

Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.