Light, oxygen, voltage (LOV) proteins, a ubiquitously distributed class of photoreceptors, regulate a wide variety of light-dependent physiological responses. Because of their modular architecture, LOV domains, i.e., the sensory domains of LOV photoreceptors, have been widely used for the construction of optogenetic tools. We recently described the structure and function of a short LOV protein (DsLOV) from the marine phototropic bacterium Dinoroseobacter shibae, for which, in contrast to other LOV photoreceptors, the dark state represents the physiologically relevant signaling state. Among bacterial LOV photoreceptors, DsLOV possesses an exceptionally fast dark recovery, corroborating its function as a "dark" sensor. To address the mechanistic basis of this unusual characteristic, we performed a comprehensive mutational, kinetic, thermodynamic, and structural characterization of DsLOV. The mechanistic basis of the fast dark recovery of the protein was revealed by mutation of the previously noted uncommon residue substitution at position 49 found in DsLOV. The substitution of M49 with different residues that are naturally conserved in LOV domains tuned the dark-recovery time of DsLOV over 3 orders of magnitude, without grossly affecting its overall structure or the light-dependent structural change observed for the wild-type protein. Our study thus provides a striking example of how nature can achieve LOV photocycle tuning by subtle structural alterations in the LOV domain active site, highlighting the easy evolutionary adaptability of the light sensory function. At the same time, our data provide guidance for the mutational photocycle tuning of LOV domains, with relevance for the growing field of optogenetics.

Introduction:Transforming growth factor β (TGFβ) promotes cell survival by endorsing DNA damage repair and mediates an immunosuppressive tumor microenvironment. Thus, TGFβ activation in response to radiation therapy is potentially targetable because it opposes therapeutic control. Strategies to assess this potential in the clinic are needed. Methods & Materials:We evaluated positron emission tomography (PET) to image 89 Zrfresolimumab, a humanized TGFβ neutralizing monoclonal antibody, as means to detect TGFβ

Background and PurposeAerobic glycolysis is a unique feature of tumour cells that entails several advantages for cancer progression such as resistance to apoptosis. The low MW compound, dichloroacetate, is a pyruvate dehydrogenase kinase inhibitor, which restores oxidative phosphorylation and induces apoptosis in a variety of cancer entities. However, its therapeutic effectiveness is limited by resistance mechanisms. This study aimed to examine the role of the anti‐apoptotic hyaluronan (HA) matrix in this context and to identify a potential add‐on treatment option to overcome this limitation.Experimental ApproachThe metabolic connection between dichloroacetate treatment and HA matrix augmentation was analysed in vitro by quantitative PCR and affinity cytochemistry. Metabolic pathways were analysed using Seahorse, HPLC, fluorophore‐assisted carbohydrate electrophoresis, colourimetry, immunoblots, and immunochemistry. The effects of combining dichloroacetate with the HA synthesis inhibitor 4‐methylumbelliferone was evaluated in 2D and 3D cell cultures and in a nude mouse tumour xenograft regression model by immunoblot, immunochemistry, and FACS analysis.Key ResultsMitochondrial reactivation induced by dichloroacetate metabolically activated HA synthesis by augmenting precursors as well as O‐GlcNAcylation. This process was blocked by 4‐methylumbelliferone, resulting in enhanced anti‐tumour efficacy in 2D and 3D cell culture and in a nude mouse tumour xenograft regression model.Conclusions and ImplicationsThe HA rich tumour micro‐environment represents a metabolic factor contributing to chemotherapy resistance. HA synthesis inhibition exhibited pronounced synergistic actions with dichloroacetate treatment on oesophageal tumour cell proliferation and survival in vitro and in vivo suggesting the combination of these two strategies is an effective anticancer therapy.