Michal
Hershfinkel
*a,
Dianne
Ford
b,
Shannon
Kelleher
c and
Elias
Aizenman
ad
aDepartment of Physiology and Cell Biology and The Zlotowski Center of Neuroscience, Ben-Gurion University of the Negev, Faculty of Health Sciences, POB 653, Beer-Sheva, 84105, Israel. E-mail: hmichal@bgu.ac.il; Fax: +972-8-6477627; Tel: +972-8-6477-318
bInstitute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle upon Tyne, UK
cDepts. of Cellular and Molecular Physiology, Pharmacology and Surgery, Penn State Hershey College of Medicine, Hershey, PA 17033, USA
dDepartment of Neurobiology and Pittsburgh Institute for Neurodegenerative Disorders, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
Zinc is an essential metal ion in all living organisms, serving not only as a catalytic and structural cofactor, but also as a first and second messenger in a large number of cellular signaling processes (Fukada et al. 2014, ref. 1). The complexity of cellular zinc regulation is indeed reflected by the presence of nearly 3000 zinc-interacting proteins, including 24 identified zinc membrane transporters. This meeting highlighted recent advances in mechanisms underlying zinc transport and signaling, which will pave the way for a better understanding of the physiological roles of this metal and the requirements for zinc as a micronutrient. Moreover, innovative analytical and chemical tools for determining cellular zinc levels, a highly debated topic in the field, were presented at this meeting. The many high impact and exciting new developments in the field of zinc biology (Fig. 1), presented at this vibrant meeting are summarized briefly in this report.
Our knowledge of cellular zinc signaling and homeostasis has lagged behind that of other metal ions due to the previous lack of zinc-specific tools, and the inability to differentiate between calcium and zinc signals within cells. Stephen J. Lippard, who has developed multiple chemical tools for tracking and controlling cellular zinc ions, elegantly described how the field of metal-binding fluorescent tools has evolved, focusing on several novel fluorescent dyes that permit discrimination of minute changes in intracellular free zinc with a high signal to noise ratio. Moreover, Lippard described the use of a new generation of ultra-fast and highly specific zinc chelators as powerful tools to understand the functions of synaptically released zinc in the auditory and olfactory systems.
Measuring zinc status in humans remains a major challenge in the field and is particularly important for studies addressing the essentiality of the metal as a nutrient. In the third Plenary Lecture, Janet C. King suggested that in the absence of a reliable biomarker, dietary zinc intake and organismal physiological measurements such as growth and immunomodulation could be used to determine adequate zinc status. King noted, however, that zinc intake recommendations are confounded by the need to account for effects of factors that inhibit its absorption, such as dietary levels of plant-derived phytates. Multidisciplinary approaches are required to advance the development of reliable assessment tools and to address the consequences of deficient or excess zinc intake on health and disease.
Finally, pioneers of zinc biology gave highly educative historical reviews of the field and challenged the audience with further questions that remain to be addressed in order to move forward in the future. Ananda S. Prasad and Harold H. Sandstead were among the first to identify the critical role of dietary zinc in growth, development and immune system function. They presented the enormous progress made in the field of zinc nutrition observed during their professional lifetime, yet highlighted the continuing need for advancing the knowledge of the impact of proper zinc status on human health. Dennis Choi, an early zinc neurobiologist who investigated the role of this metal in neurodegenerative disorders, highlighted the urgent need for accelerating translation of basic science observations into clinical practice and encouraged the audience, especially the younger participants, to ardently continue to tackle difficult problems with their research.
All keynote lectures showcased the rapid growth of the field and how mechanistic details are being elucidated to give a sound understanding of the role of zinc in biological systems. Although a consensus emerged that we are now seeing beyond the tip of the biological zinc iceberg, we have yet to uncover many pathways that will lead to understanding the role of the metal in living organisms.
The ISZB awarded the Frederickson Prize for seminal studies in the field of zinc biology to John H. Weiss. In his prize acceptance presentation, Weiss described major advances that lead to elucidating the role zinc plays in the central nervous system. The initial description of a releasable pool of zinc ions in synaptic vesicles by Gorm Danscher and Christopher J. Frederickson was the trigger that instigated his own studies on this topic. Weiss described his work that identified a role for calcium-permeable AMPA channels as a major permeation pathway for entry of synaptically released zinc into post-synaptic neurons. He then presented his studies on the intracellular release of zinc from mitochondria, accompanied by the generation of reactive oxygen species and increased neuronal cell death during ischemic injury.
The large number of Zn2+ transporters present in the mammalian cell raises numerous questions regarding their precise roles, a subject that was discussed in several sessions throughout the meeting. For example, Johannes Engelken raised a general hypothesis that changes in Zn2+ transporter expression is a driving force underlying carcinogenesis. Yehuda G. Assaraf showed how ZnT transporters form both, homo- and hetero-dimers using a bimolecular fluorescence complementation (BiFC) technique. A physiological role for such dimerization had been previously described by Gloria Salazar, who presented new results on ZnT10 dimerization and its role in protein localization. Taiho Kambe reported a role for the coordinated action of ZnT transporters and metallothioneins (MTs), required for the activation of Zn2+-dependent enzymes. Miki Kawachi, Dax Fu and Robert E. Dempski showed how putative Zn2+ binding sites on various transporters affect the kinetics of ion transport and thereby regulate physiological functions, such as metal tolerance of cells.
Using Drosophila melanogaster, Richard Burke dissected the roles of various members of the ZnT family in conditions of either zinc excess or deficiency through their effect on the proper development of the fly eye. Fanis Missirlis expanded the analysis to show how excess zinc is stored to protect either individual cells or the whole organism from zinc toxicity. The same Drosophila model was used by David W. Killilea to identify a role of zinc transporters in urinary stone formation and tubule obstruction. He further showed that this effect was dependent on dietary zinc levels, linking transporter function and excess zinc to the formation of urinary stones. Using Caenorhabditis elegans as a model, S. Kerry Kornfeld has identified novel genes that are involved in zinc metabolism and are induced by excess zinc. Christer Hogstrand used the zebrafish model to elegantly show the importance of ZIP and ZnT transporters in development, in particular ZIP10.
Attesting to their importance, ZIP proteins are widely expressed across species and preserved in evolution. Gerold Schmitt-Ulms described his finding that the prion gene family descended from an ancestral Liv-ZIP gene, and maintained structural similarity with the Zn2+ transporter. His study further revealed a role for ZIP6 and the prion protein in the epithelial/mesenchymal transition. Nigel M. Hooper discussed a different angle of Zn2+-related prion protein physiology by showing how neuronal Zn2+ accumulation is enhanced by prion proteins and linked to amyloid-beta oligomer signaling.
Several studies have described the “hijacking” of zinc transporters by other metal ions such as manganese, iron and cadmium. In this context and using Drosophila melanogaster as a model, Guiran Xiao showed a role for ZIP13 in iron transport and suggested that in Ehlers-Danlos syndrome, in which hZIP13 is defective, iron dyshomeostasis, in addition to zinc, may be responsible for some of the observed pathology. Similarly, Michael D. Knutson described a role for ZIP14 in iron transport in the liver, and suggested that modulation of ZIP14 expression is related to hemochromatosis. Collectively, these investigations implicate a role for zinc in modulating the activity of both environmental metals and other biometals.
Jeanne A. Hardy presented a study of the binding of Zn2+ to distinct sites on caspases and the role of this process in caspase-initiated apoptosis. Another example for a role of Zn2+-binding in regulation of physiological function was presented by Deborah B. Zamble, who described a regulatory metal binding site on members of a subfamily of P-loop GTPases. Kengo Homma presented results indicating that Zn2+-deficiency induces conformational changes in the Zn2+ binding protein SOD1 and subsequent ER stress, similar to the effects of SOD1-mutants associated with amyotrophic lateral sclerosis (ALS). In the blood Zn2+ is bound to albumin, Alan Stewart and Claudia A. Blindauer identified the binding site for this ion and showed that this site may be disrupted by other metal ions or fatty acids, an issue that may link the metabolism of these essential nutrients.
Zinc binding by microbes is essential for their survival and function, and thus Zn2+ binding proteins were also discussed in relation to host/pathogen interactions. Calprotectin, a protein with antimicrobial functions, was shown to have Zn2+ binding sites. Elizabeth M. Nolan further described how these sites are regulated by Ca2+, suggesting a tunable affinity for Zn2+. Thus upon its release, calprotectin is thought to compete with pathogens for available Zn2+ ions. Megan B. Brophy then suggested that S100 Ca2+ binding proteins may have a role in Zn2+ sequestration and antimicrobial effects. In the gut, Zn2+ is available for absorption; yet, during inflammatory conditions the levels of this ion are regulated by the release of the Zn2+ binding protein calprotectin. Expression of a high affinity Zn2+ transporter by the food borne pathogen Salmonella enterica serovar Typhimurium was described by Manuela Raffatellu. This transporter enhances the resistance of the bacteria to Zn2+. Raffatellu further discussed how intestinal microbes compete for Zn2+ ions in the inflamed gut and limit zinc availability to pathogens, thereby protecting the intestine. A specific role for macrophages in this process was discussed by Mathew J. Sweet, who showed upregulation of Zn2+ transporters in these cells following exposure to bacteria.
As the tight regulation of cellular Zn2+ levels is essential for all living species, the role of transcription factors and proteins regulated by Zn2+ may also have common features across phylogeny. Dianne Ford presented recent work that supports the theory that Zn2+ initially regulates MT and ZnT1 through MTF1, and that the transcription of secondary processes to maintain Zn2+ levels within cells is activated when this buffering capacity is exceeded. She also presented observations revealing that ZNF658, a newly identified zinc-sensitive transcription factor, plays a role in coordinating zinc homeostasis with ribosome biogenesis. Amanda J. Bird described how a yeast transcription factor, Loz1, is regulated by Zn2+ to control intracellular Zn2+ distribution and protect yeast cells from Zn2+ toxicity. David J. Eide described the role of another transcription factor that is regulated by Zn2+ in yeast, Zap1, and its target gene, Tsa1, in dysregulation of protein folding under zinc deficiency. Wolfgang Wohlleben showed that the bacterial Zur gene is a Zn2+ sensor that regulates the production of the zincophore EDDS (ethylenediaminedisuccinic acid), and knockdown of this gene allowed high yield of EDDS production that was independent of Zn2+. This may have applications in industry by allowing replacement of EDTA, a toxic non-biodegradable pollutant, with EDDS.
Epithelial cell physiology is becoming an important field for Zn2+ signaling research. Changes in zinc concentrations or in Zn2+ transporters are particularly apparent in breast cells, during normal hormonal cycles as well as during lactation, involution or even in cancer. Shannon L. Kelleher presented results from a comprehensive study on the role of ZnT2 in lactation and in regulating the involution of the tissue after lactation and the consequences of naturally occurring genetic variants. Absorption of dietary Zn2+ is done by epithelial intestinal cells expressing ZIP4 and ZIP5 proteins, and their expression is highly regulated by this ion. Novel results, however, by Agnes Michalczyk suggest that ZIP1 is expressed on the apical microvilli of the Caco-2 colonocyte cells, and is also involved in regulation of Zn2+ accumulation. Intestinal epithelial cells express a metabotropic Zn2+-sensing receptor, ZnR/GPR39, which triggers Zn2+-dependent signaling. Michal Hershfinkel described a role for the ZnR/GPR39 in modulating ion transport function as well as the formation of the tight junction barriers in the colon epithelial cells. These results further suggest that ZnR/GPR39 may be important in inflammatory bowel disease as well as diarrhea. A role for Zn2+ released from mast cells was presented by Keigo Nishida who linked Zn2+-dependent signaling to release of cytokines and wound healing.
Specific physiological roles for vesicular Zn2+ have been a major issue of debate in the field for several years. A particular issue is centered on the fact that ZnT knockout mouse models result in a limited phenotype. One such example is the ZnT3 knockout mice, devoid of vesicular zinc, which appear to have normal neuronal function. Changing this notion, Richard H. Dyck showed that activity-dependent hippocampal neurogenesis is impaired in the ZnT3 deficient mice. This was further related to impairment of hippocampal-dependent behavioral tasks in the ZnT3 deficient animals. A role for Zn2+ in neurogenesis following traumatic brain injury was described by Cathy W. Levenson who identified transcription factors regulating this process in neuronal stem cells. Further supporting a role for synaptic Zn2+ in learning and memory function, Haruna Tamano, Atsushi Takeda and Yang Li presented results from electrophysiological studies of long term potentiation, LTP, linking synaptic Zn2+ release to the regulation of neuronal activity in the hippocampus. Richard L. Chappell suggested that synaptic Zn2+ released from neurons has a feedback role in regulating neuronal response to a very wide range of stimuli in the retina. Using a multiple sclerosis model, Sang Won Suh revealed changes in ZnT3 expression upon induction of the demyelination process. His results thus link ZnT3 and neuronal vesicular zinc to this serious human disease. Paul T. Francis, using tissue from human patients, showed that loss of the same zinc transporter is associated with Lewy body dementia.
Another interesting aspect of neuronal Zn2+ is the association with neurological disorders. For example, Shank scaffold proteins localized to postsynaptic sites seem to be correlated with overall zinc levels in patients with autism, as demonstrated by Andreas M. Grabrucker. Another psychiatric disease sometimes linked to zinc deficiency is schizophrenia, and Elizabeth Scarr showed changes in ZIP12 mRNA levels in patients with this serious neurobehavioral disorder. This protein was also shown by Winyoo Chowanadisai to regulate neurite outgrowth and neuronal differentiation via a CREB-dependent pathway. A mutation in PARK9 ATPase causes Parkinsonism, and was shown by Taiji Tsunemi to render the cells susceptible to Zn2+ toxicity, suggesting that lowering Zn2+ may be beneficial to patients with this disease. Recent developments concerning the importance of zinc in Alzheimer's disease, from the formation of Aβ oligomers to Tau phosphorylation, which has been an important topic at previous ISZB meetings, were presented. Jae-Young Koh suggested a role for Zn2+-dependent lysosomal permeabilization in this disease and Bing Zhou suggested a role for Zn2+ in Tau phosphorylation. Ashley I. Bush described the complexity of applying the tools developed through basic research to therapeutics, via the example of the Zn2+ ionophore, PBT2. This compound was tested on animal models and shown to decrease Aβ load, restore zinc homeostasis and improve cognitive function.
The ISZB would like to thank Qiagen, Pfizer, Teva Pharmaceutical Industries, Novartis Pharma AG, Shino Test Corporation, Strem Chemicals, Genostaff and Hamari Chemicals for their generous donations that helped support the meeting. Book prizes for excellent talks were kindly provided by The Royal Society of Chemistry, Springer and IOS Press. Travel awards were supported in part by NIH grant GM112419. The authors would also like to thank Wolfgang Maret, Daren L. Knoell, Robert A. Colvin and David I. Soybel for their invaluable contributions to the organization of the meeting.
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