Copper isotope ratios in serum do not track cancerous tumor evolution, but organ failure.

Relative to healthy controls, lighter copper isotopic compositions have been observed in the serum of breast cancer and end-stage liver disease patients, raising the possibility that Cu isotope ratios could be used as a tracer for disease progression. Here, we assess the potential of natural Cu isotopic variations (expressed as δ65Cu) as diagnostic tools for cancer progression and/or liver failure by performing a first-order analysis of Cu isotopic cycling in the human body. Using a box model, we simulate the kinetics of Cu mass transfer throughout significant reservoirs in the body, allowing isotopic fractionation to occur during Cu uptake/release from these reservoirs. With this model, we determine under which conditions the serum δ65Cu values would reflect perturbation related to cancer growth and/or liver failure at a level resolvable with modern mass spectrometry. We find that tumor growth alone is unable to explain the light isotopic signature observed in serum. Instead, we find that metabolic changes to the liver function resulting in a ∼1‰ isotope fractionation during Cu uptake from the blood into the liver can readily explain the long-term serum isotopic shift of ∼0.2‰ observed in cancer patients. A similar fractionation (∼1.3‰) during Cu uptake into the liver also readily explains the -1.2‰ shift observed in the serum of cirrhosis patients with ascites, suggesting a potentially common driver of isotopic fractionation in both cases. Using this model, we then test hypotheses put forward by previous studies and begin to probe the mechanisms behind the measured isotopic compositions.

Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in the regulation of apoptosis, proliferation, and DNA-damage response, is overexpressed in many cancers, including small cell lung cancer (SCLC). Here we report that LB100, a small molecule inhibitor of PP2A, when combined with platinum-based chemotherapy, synergistically elicited an antitumor response both in vitro and in vivo with no apparent toxicity. Using inductively coupled plasma mass spectrometry, we determined quantitatively that sensitization via LB100 was mediated by increased uptake of carboplatin in SCLC cells. Treatment with LB100 alone or in combination resulted in inhibition of cell viability in two-dimensional culture and three-dimensional spheroid models of SCLC, reduced glucose uptake, and attenuated mitochondrial and glycolytic ATP production. Combining LB100 with atezolizumab increased the capacity of T cells to infiltrate and kill tumor spheroids, and combining LB100 with carboplatin caused hyperphosphorylation of the DNA repair marker γH2AX and enhanced apoptosis while attenuating MET signaling and invasion through an endothelial cell monolayer. Taken together, these data highlight the translational potential of inhibiting PP2A with LB100 in combination with platinum-based chemotherapy and immunotherapy in SCLC.

A Disorder-to-Order Transition Activates an ATP-Independent Membrane Protein Chaperone.

The 43 kDa subunit of the chloroplast signal recognition particle, cpSRP43, is an ATP-independent chaperone essential for the biogenesis of the light harvesting chlorophyll-binding proteins (LHCP), the most abundant membrane protein family on earth. cpSRP43 is activated by a stromal factor, cpSRP54, to more effectively capture and solubilize LHCPs. The molecular mechanism underlying this chaperone activation is unclear. Here, a combination of hydrogen-deuterium exchange, electron paramagnetic resonance, and NMR spectroscopy experiments reveal that a disorder-to-order transition of the ankyrin repeat motifs in the substrate binding domain of cpSRP43 drives its activation. An analogous coil-to-helix transition in the bridging helix, which connects the ankyrin repeat motifs to the cpSRP54 binding site in the second chromodomain, mediates long-range allosteric communication of cpSRP43 with its activating binding partner. Our results provide a molecular model to explain how the conformational dynamics of cpSRP43 enables regulation of its chaperone activity and suggest a general mechanism by which ATP-independent chaperones with cooperatively folding domains can be regulated.

Two distinct sites of client protein interaction with the chaperone cpSRP43.

Integral membrane proteins are prone to aggregation and misfolding in aqueous environments and therefore require binding by molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is an ATP-independent chaperone required for the biogenesis of the most abundant class of membrane proteins, the light-harvesting chlorophyll a/b-binding proteins (LHCPs). Previous work has shown that cpSRP43 specifically recognizes an L18 loop sequence conserved among LHCP paralogs. However, how cpSRP43 protects the transmembrane domains (TMDs) of LHCP from aggregation was unclear. In this work, alkylation-protection and site-specific cross-linking experiments found that cpSRP43 makes extensive contacts with all the TMDs in LHCP. Site-directed mutagenesis identified a class of cpSRP43 mutants that bind tightly to the L18 sequence but are defective in chaperoning full-length LHCP. These mutations mapped to hydrophobic surfaces on or near the bridging helix and the ß-hairpins lining the ankyrin repeat motifs of cpSRP43, suggesting that these regions are potential sites for interaction with the client TMDs. Our results suggest a working model for client protein interactions in this membrane protein chaperone.