David Carmena

Structure of mammalian AMPK and its regulation by ADP

The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.

Structural basis of AMPK regulation by small molecule activators

AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

ADP Regulates SNF1, the Saccharomyces cerevisiae Homolog of AMP-Activated Protein Kinase

Summary The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.

A review of the global prevalence, molecular epidemiology and economics of cystic echinococcosis in production animals

Cystic echinococcosis (CE) is an important and widespread zoonotic infection caused by the larval stages of taeniid cestodes of the genus Echinococcus. The disease represents a serious animal health concern in many rural areas of the world, causing important economic losses derived from decreased productivity and viscera condemnation in livestock species. In this review we aim to provide a comprehensive overview on recent research progress in the epidemiology of CE in production animals from a global perspective. Particular attention has been paid to the discussion of the extent and significance of recent molecular epidemiologic data. The financial burden associated to CE on the livestock industry has also been addressed. Data presented are expected to improve our current understanding of the parasites geographical distribution, transmission, host range, immunogenicity, pathogenesis, and genotype frequencies. This information should be also valuable for the design and implementation of more efficient control strategies against CE.

Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

Aims:  To evaluate the prevalence of Cryptosporidium and Giardia in surface water supplies from the province of Álava, northern Spain, and to investigate possible associations among the presence of these pathogenic protozoa with microbiological, physicochemical and atmospheric parameters.

Analysis of the economic impact of cystic echinococcosis in Spain

OBJECTIVE To estimate the overall economic losses due to human and animal cystic echinococcosis (CE) in Spain in 2005. METHODS We obtained data on annual CE incidence from surveillance and abattoir records, and on CE-related treatment and productivity losses (human and animal) from the scientific literature. Direct costs were those associated with diagnosis, surgical or chemotherapeutic treatment, medical care and hospitalization in humans, and condemnation of offal in livestock (sheep, goats, cattle and pigs). Indirect costs comprised human productivity losses and the reduction in growth, fecundity and milk production in livestock. The Latin hypercube method was used to represent the uncertainty surrounding the input parameters. FINDINGS The overall economic loss attributable to CE in humans and animals in 2005 was estimated at 148 964 534 euros (euro) (95% credible interval, CI: 21 980 446-394 012 706). Human-associated losses were estimated at euro133 416 601 (95% CI: 6 658 738-379 273 434) and animal-associated losses at euro15 532 242 (95% CI: 13 447 378-17 789 491). CONCLUSION CE is a neglected zoonosis that remains a human and animal health concern for Spain. More accurate data on CE prevalence in humans (particularly undiagnosed or asymptomatic cases) and better methods to estimate productivity losses in animals are needed. CE continues to affect certain areas of Spain, despite several control initiatives since 1986. Given the high economic burden of CE, additional funding is needed to reduce human and animal infection rates through improved disease surveillance, regular treatment of dogs and greater cooperation between agencies.

Echinococcus granulosus infection in Spain.

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus is an endemic disease in Spain. Although specific control programmes initiated in the 1980s have led to marked reductions in CE infection rates in Spain, the disease still remains an important human and animal health problem in many regions of the country. Human incidence and livestock (including sheep, cattle, pigs and horses) prevalence data were gathered from national epidemiological surveillance information systems and regional institutions for the period 2000–2005. Additionally, data on the prevalence of E. granulosus infection in dogs were obtained from published literature. The most affected regions were those of the North Eastern, Central and Western parts of the country, (Autonomous Regions of Aragon, Castile‐La Mancha, Castile‐Leon, Extremadura, Navarre and La Rioja), where human CE incidence rates in the range of 1.1–3.4 cases per 105 inhabitants coexist with ovine/bovine CE prevalence rates up to 23%. Control programmes of hydatidosis/echinococcosis should be reinforced in these regions to reduce the prevalence of the disease.

Canine echinococcosis: global epidemiology and genotypic diversity.

Canine echinococcosis is a potential zoonotic infection caused by the adult form of several cestode species belonging to the genus Echinococcus, of which E. granulosus sensu lato and E. multilocularis are the most epidemiologically relevant. Dogs infected with E. granulosus and E. multilocularis are widely regarded as the main source of infection for human cystic and alveolar echinococcosis, diseases that cause substantial morbidity and socio-economic burden in several regions of the world. Following our previous review on the global situation of cystic echinococcosis in livestock species (Cardona and Carmena. Vet. Parasitol. 2013;192:10-32), we summarize here current knowledge on the global epidemiology, geographical distribution and molecular diversity of Echinococcus spp. infection in dogs. We address relevant topics including the implications of the increasing urbanization of wildlife species such as foxes, coyotes, and dingoes in the establishment of urban cycles of Echinococcus spp., or the rising concerns regarding the role of unsupervised translocation of infected dogs in spreading the infection to Echinococcus-free areas. The involvement of wildlife species as natural reservoirs of disease to domestic animals and humans and the epidemiological significance of the sympatric occurrence of different Echinococcus species in the same geographical region are also debated. Data presented are expected to be useful for policy makers, educational and health authorities responsible for designing and implementing effective measures for disease control and prevention.

ClC-3 expression enhances etoposide resistance by increasing acidification of the late endocytic compartment

Resistance to anticancer drugs and consequent failure of chemotherapy is a complex problem severely limiting therapeutic options in metastatic cancer. Many studies have shown a role for drug efflux pumps of the ATP-binding cassette transporters family in the development of drug resistance. ClC-3, a member of the CLC family of chloride channels and transporters, is expressed in intracellular compartments of neuronal cells and involved in vesicular acidification. It has previously been suggested that acidification of intracellular organelles can promote drug resistance by increasing drug sequestration. Therefore, we hypothesized a role for ClC-3 in drug resistance. Here, we show that ClC-3 is expressed in neuroendocrine tumor cell lines, such as BON, LCC-18, and QGP-1, and localized in intracellular vesicles colabeled with the late endosomal/lysosomal marker LAMP-1. ClC-3 overexpression increased the acidity of intracellular vesicles, as assessed by acridine orange staining, and enhanced resistance to the chemotherapeutic drug etoposide by almost doubling the IC50 in either BON or HEK293 cell lines. Prevention of organellar acidification, by inhibition of the vacuolar H+-ATPase, reduced etoposide resistance. No expression of common multidrug resistance transporters, such as P-glycoprotein or multidrug-related protein-1, was detected in either the BON parental cell line or the derivative clone overexpressing ClC-3. The probable mechanism of enhanced etoposide resistance can be attributed to the increase of vesicular acidification as consequence of ClC-3 overexpression. This study therefore provides first evidence for a role of intracellular CLC proteins in the modulation of cancer drug resistance. [Mol Cancer Ther 2007;6(3):979–86]

Identification and molecular characterization of Cryptosporidium and Giardia in children and cattle populations from the province of Álava, North of Spain.

The prevalence of and factors associated with the protozoan enteropathogens Cryptosporidium and Giardia have been investigated in selected children and cattle populations from the province of Álava (Northern Spain). The presence of these organisms was detected in fecal samples using commercially available coproantigen-ELISA (CpAg-ELISA) and immunochromatographic (ICT) assays. A total of 327 caregivers of children participants were asked to answer questions on risk factors potentially associated to the prevalence of Cryptosporidium and Giardia, including water-use practices, water sports and contact with domestic or pet animals. Molecular analyses were conducted using a nested-PCR technique to amplify the small-subunit (SSU) rRNA gene of Cryptosporidium and the triosephosphate isomerase (tpi) gene of Giardia. Cryptosporidium oocysts and Giardia cysts were found in 3 and 16 samples using the CpAg-ELISA, and in 5 and 9 samples using the ICT test, respectively. Cryptosporidium and Giardia were also found in 7 and 17 samples by CpAg-ELISA, and 4 and 14 samples by ICT, respectively, of 227 cattle fecal samples. The overall Cryptosporidium and Giardia infection prevalences, based on a Bayesian approach accounting for the imperfect sensitivities and specificities of both diagnostic tests, were estimated to 1.0% (95% BCI: 0.2%-2.8%) and 3.1% (1.5%-5.3%) in children and 3.0% (0.5%-9.2%) and 1.4% (0.0%-6.4%) in cattle, respectively. In humans, a single Cryptosporidium isolate was characterized as C. hominis. Of seven Giardia isolates, four were identified as assemblage B, two as assemblage A-II and one was a mixed assemblage B+A-II infection. No Cryptosporidium or Giardia isolates could be obtained from cattle samples. Although limited, these results seem to suggest that cattle are unlikely to be an important reservoir of zoonotic Cryptosporidium and/or Giardia in the province of Álava.