Two-compartment tumor metabolism: Autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells

Previously, we proposed a new paradigm to explain the compartment-specific role of autophagy in tumor metabolism. In this model, autophagy and mitochondrial dysfunction in the tumor stroma promotes cellular catabolism, which results in the production of recycled nutrients. These chemical building blocks and high-energy “fuels” would then drive the anabolic growth of tumors, via autophagy resistance and oxidative mitochondrial metabolism in cancer cells. We have termed this new form of stromal-epithelial metabolic coupling: “two-compartment tumor metabolism.” Here, we stringently tested this energy-transfer hypothesis, by genetically creating (1) constitutively autophagic fibroblasts, with mitochondrial dysfunction or (2) autophagy-resistant cancer cells, with increased mitochondrial function. Autophagic fibroblasts were generated by stably overexpressing key target genes that lead to AMP-kinase activation, such as DRAM and LKB1. Autophagy-resistant cancer cells were derived by overexpressing GOLPH3, which functionally promotes mitochondrial biogenesis. As predicted, DRAM and LKB1 overexpressing fibroblasts were constitutively autophagic and effectively promoted tumor growth. We validated that autophagic fibroblasts showed mitochondrial dysfunction, with increased production of mitochondrial fuels (L-lactate and ketone body accumulation). Conversely, GOLPH3 overexpressing breast cancer cells were autophagy-resistant, and showed signs of increased mitochondrial biogenesis and function, which resulted in increased tumor growth. Thus, autophagy in the tumor stroma and oxidative mitochondrial metabolism (OXPHOS) in cancer cells can both dramatically promote tumor growth, independently of tumor angiogenesis. For the first time, our current studies also link the DNA damage response in the tumor microenvironment with “Warburg-like” cancer metabolism, as DRAM is a DNA damage/repair target gene.     

Diagnosing Severe Falciparum Malaria in Parasitaemic African Children: A Prospective Evaluation of Plasma PfHRP2 Measurement

Background

In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria.

Methods and Findings

Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350–1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991–1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log₁₀ plasma PfHRP2 and risk of death. Mortality increased 20% per log₁₀ increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05–1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44–0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69–1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings.

Conclusions

Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of “true” severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children. Please see later in the article for the Editors'' Summary.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

桶形芋螺毒液蛋白质组分析及二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶。在热带药用海洋生物芋螺的毒液中,富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内氧化折叠至关重要。研究上样量、水化方式与时间、除盐步骤、聚焦电压和时间、平衡时间、凝胶电泳方法和染色方法等方面,优化并建立了较为理想的芋螺毒液蛋白样品双向电泳的实验条件与方法;通过双向电泳和MALDI-TOF-MS质谱分析技术,从海南产桶形芋螺(Conus betulinus Linnaeus)毒液蛋白中成功分离鉴定出PDI等9种蛋白;通过双向电泳和PDQuest软件分析了并比较了三种不同大小桶形芋螺毒液总蛋白的差异性,并质谱鉴定出5个明显的差异蛋白点。研究建立了桶形芋螺毒液蛋白双向电泳分析及质谱鉴定的技术方法,为后续大量分离纯化出天然的芋螺PDI酶以及利用蛋白质组学技术方法深入研究芋螺毒液特征提供了重要的基础。     

The twin-arginine translocation (Tat) protein export pathway

The twin-arginine translocation (Tat) protein export system is present in the cytoplasmic membranes of most bacteria and archaea and has the highly unusual property of transporting fully folded proteins. The system must therefore provide a transmembrane pathway that is large enough to allow the passage of structured macromolecular substrates of different sizes but that maintains the impermeability of the membrane to ions. In the Gram-negative bacterium Escherichia coli, this complex task can be achieved by using only three small membrane proteins: TatA, TatB and TatC. In this Review, we summarize recent advances in our understanding of how this remarkable machine operates.     

Protein conducting channels-mechanisms, structures and applications

In the past decade among the main developments in the field of bionanotechnology is the application of proteins in devices. Research focuses on the modification of enzyme systems by means of chemical and physical tools in order to achieve full control of their function and to employ them for specific tasks. Membrane protein channels are intriguing biological devices as they allow the recognition and passage of a variety of macromolecules through an otherwise impermeable lipid bilayer. Hence, membrane proteins can be used as sensory devices for detection or as molecular nanovalves to allow for the controlled release of molecules. Here, we discuss the structure and function of three different channel proteins that mediate the membrane passage of macromolecules using different mechanisms. These systems are described in a comparative manner and an overview is provided of the technological advances in employing these proteins in external (or human) controllable devices.     

Founder mutations in Tunisia: implications for diagnosis in North Africa and Middle East

Cystinuria (OMIM 220100) is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones. Among the heterogeneous group of kidney stone diseases, cystinuria is the only disorder which is exclusively caused by gene mutations. So far, two genes responsible for cystinuria have been identified: SLC3A1 (chromosome 2p21) encodes the heavy subunit rBAT of a renal b^0,+ transporter while SLC7A9 (chromosome 19q12) encodes its interacting light subunit b^0,+AT. Mutations in SLC3A1 are generally associated with an autosomal-recessive mode of inheritance whereas SLC7A9 variants result in a broad clinical variability even within the same family. The detection rate for mutations in these genes is larger than 85%, but it is influenced by the ethnic origin of a patient and the pathophysiological significance of the mutations. In addition to isolated cystinuria, patients suffering from the hypotonia-cystinuria syndrome have been reported carrying deletions including at least the SLC3A1 and the PREPL genes in 2p21. By extensive molecular screening studies in large cohort of patients a broad spectrum of mutations could be identified, several of these variants were functionally analysed and thereby allowed insights in the pathology of the disease as well as in the renal trafficking of cystine and the dibasic amino acids. In our review we will summarize the current knowledge on the physiological and the genetic basis of cystinuria as an inborn cause of kidney stones, and the application of this knowledge in genetic testing strategies.