Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Metabolomics in Cell Biology.

Metabolomics has long been used in a biomedical context. The most typical samples are body fluids in which small molecules can be detected and quantified using technologies such as Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Many studies, in particular in the wider field of cancer research, are based on cellular models. Different cancer cells can have vastly different ways of regulating metabolism and responses to drug treatments depend on specific metabolic mechanisms which are often cell type specific. This has led to a series of publications using metabolomics to study metabolic mechanisms. Cell-based metabolomics has specific requirements and allows for interesting approaches where metabolism is followed in real-time. Here applications of metabolomics in cell biology have been reviewed, providing insight into specific technologies used and showing exemplary case studies with an emphasis towards applications which help to understand drug mechanisms.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL).

BL and DLBCL are subtypes of B-cell lymphomas that arise from germinal centre B lymphocytes. Differentiation between BL and DLBCL is critical and can be challenging, as these two types of cancer share the same morphological, immunophenotypic, and genetic characteristics. In this study, we have examined metabolism in BL and DLBCL lymphomas and found distinctive differences in serine metabolism. We show that BL cells consume significantly more extracellular asparagine than DLBCL cells. Using a tracer-based approach, we find that asparagine regulates the serine uptake and serine synthesis in BL and DLBCL cells. Calculation of Differentially Expressed Genes (DEGs) from RNAseq datasets of BL and DLBCL patients show that BL cancers express the genes involved in serine synthesis at a higher level than DLBCL. Remarkably, combined use of an inhibitor of serine biosynthesis pathway and an anticancer drug asparaginase increases the sensitivity of BL cells to extracellular asparagine deprivation without inducing a change in the sensitivity of DLBCL cells to asparaginase. In summary, our study unravels metabolic differences between BL and DLBCL with diagnostic potential which may also open new avenues for treatment.

A framework for tracer-based metabolism in mammalian cells by NMR.

Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation. However, for applications using human cells and cell lines, the challenge is to optimize the process to maximize sensitivity and reproducibility. Here we present a new framework to analyze metabolism in mammalian cell lines and primary cells, covering the workflow from the preparation of cells to the acquisition and analysis of NMR spectra. We have applied this new approach in hematological and liver cancer cell lines and confirm the feasibility of tracer-based metabolism in primary liver cells.