Discovery of new gene that stimulates the release of calcium in cells

International research collaborators have identified a new family of proteins, TPC2 (two-pore channels), that facilitates calcium signaling from specialized subcellular organelles.

The study, published today in Nature , is the first to isolate TPC2 as a channel that binds to nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), a second-signaling messenger, resulting in the release of calcium from intracellular stores. According to the researchers, this new discovery may have broad implications in cell biology and human disease research.

“The discovery was the result of many researchers working as one international team toward a unified outcome. We are very appreciative of all the collaborators’ efforts,” said Jianjie Ma, PhD, professor of physiology and biophysics at UMDNJ-Robert Wood Johnson Medical School. “We are proud to be part of a study that will stand as the foundation for further exploration of human disease, helping researchers to better understand how calcium contributes to cell growth and disorders, including aging-related cardiac disease, diabetes, lysosomal cell dysfunction and the metastasis of cells in cancer.”

According to the researchers, the mechanism for how NAADP triggers the release of calcium, as well as the specific sites of calcium store targeted for release, were previously unknown. These findings indicate that NAADP, through its interaction with TPC2, targets a specific store of calcium in lysosomes, a specialized subunit within the cell that contain digestion enzymes and regulate cell function.

The study was a collaboration of investigative teams at four universities, including the laboratory of Dr. Michael Zhu at the Ohio State University, the laboratory of Dr. A. Mark Evans at the University of Edinburgh and the laboratory of Dr. Antony Galione at the University of Oxford.

The research was supported by grants from the United Kingdom’s Wellcome Trust and the British Heart Foundation, the United States’ National Institutes of Health, and the American Heart Association.

UMDNJ-ROBERT WOOD JOHNSON MEDICAL SCHOOL
As one of the nation’s leading comprehensive medical schools, Robert Wood Johnson Medical School of the University of Medicine and Dentistry of New Jersey is dedicated to the pursuit of excellence in education, research, health care delivery, and the promotion of community health. In cooperation with Robert Wood Johnson University Hospital, the medical school’s principal affiliate, they comprise New Jersey’s premier academic medical center. In addition, Robert Wood Johnson Medical School has 34 hospital affiliates and ambulatory care sites throughout the region.

New information on molecular biology of KRas protein

KRas is one of the usual suspects in cancer. It is a protein that is mutated in 30% of human tumors and has been implicated in the regulation of many cell signalling pathways.

For this reason, it is one of the main focuses of attention of international basic research and it is difficult to publish new information relating to its molecular biology. Researchers from the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the University of Barcelona (UB) have discovered a new breakdown pathway for this protein. The results, which could mean a new form of signalling involving KRas, were published in the latest edition of the Journal of Cell Biology (184(6):863-79), where they merited an appearance on the front page of the journal and an editorial comment. This study is part of the doctoral thesis of Dr. Albert Lu and includes the participation of Dr. Oriol Bachs , Dr. Carles Enrich , Dr. Neus Agell and Dr. Francesc Tebar , researchers from IDIBAPS and the Department of Cell Biology, Immunology and Neuroscience of the Faculty of Medicine of the University of Barcelona. Researchers from the University of Kyoto also took part in the study.

The article describes how the KRas protein is actively transported from the cell membrane, where most of its known activity takes place, to the lysosomes. The lysosomes are organelles responsible for breaking down proteins; this breakdown pathway was unknown in the case of KRas. Thanks to videomicroscopy techniques using a confocal microscope and the fluorescence, resonance energy transfer (FRET) technique, the researchers have observed how the protein is brought inside the cell and transported to the lysosomes. The protein remains active during this journey through the interior of the cell, which leads to the suspicion that it continues to exercise its influence on signalling pathways relating to cell proliferation and the appearance of cancers.

The signalling pathways activated by KRas are highly complex. With the newly available data, it will be necessary to investigate whether the signals emitted on the way to the lysosomes have a different meaning for the cell than those generated from the membrane, the protein’s usual site of action. These results provide clues to stimulating the elimination of KRas, a line of research that might result in new therapeutic strategies against cancer and diseases in which the formation of lysosomes is abnormal, such as Niemann-Pick disease. KRas is already used in the diagnosis of diseases such as colon, lung and breast cancer. The better we understand its biology, the more we will know about how it appears and how this and other diseases can be combatted.

http://www.idibaps.ub.edu/

The effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability.

Human lysosomal enzymes acid-beta-glucosidase (GCase) and acid-alpha-galactosidase (alpha-Gal A) hydrolyze, respectively, the sphingolipids glucosyl- and globotriaosyl- ceramide, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease. We have investigated the structure and stability of GCase and alpha-Gal A at the neutral-pH environment reflective of the endoplasmic reticulum and the acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and pH 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using alpha-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of alpha-Gal A with DGJ. Both GCase and alpha-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the GCase/IFG complex stability is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ- dependent conformational changes in alpha-Gal A are not seen. Thermodynamic parameters obtained from alpha-Gal A unfolding indicate two-state, van’t-Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and alpha-Gal A are thermodynamically stabilized by iminosugars, and suggest strategies for the development of new pharmacological chaperones for lysosomal storage disorders.

PMID: 19374450 [PubMed - as supplied by publisher]