Intracellular microbial rhodopsin-based optogenetics to control metabolism and cell signaling.

Microbial rhodopsin (MRs) ion channels and pumps have become invaluable optogenetic tools for neuroscience as well as biomedical applications. Recently, MR-optogenetics expanded towards subcellular organelles opening principally new opportunities in optogenetic control of intracellular metabolism and signaling via precise manipulations of organelle ion gradients using light. This new optogenetic field expands the opportunities for basic and medical studies of cancer, cardiovascular, and metabolic disorders, providing more detailed and accurate control of cell physiology. This review summarizes recent advances in studies of the cellular metabolic processes and signaling mediated by optogenetic tools targeting mitochondria, endoplasmic reticulum (ER), lysosomes, and synaptic vesicles. Finally, we discuss perspectives of such an optogenetic approach in both fundamental and applied research.

Light-Driven Sodium Pump as a Potential Tool for the Control of Seizures in Epilepsy.

The marine flavobacterium Krokinobactereikastus light-driven sodium pump (KR2) generates an outward sodium ion current under 530 nm light stimulation, representing a promising optogenetic tool for seizure control. However, the specifics of KR2 application to suppress epileptic activity have not yet been addressed. In the present study, we investigated the possibility of KR2 photostimulation to suppress epileptiform activity in mouse brain slices using the 4-aminopyrindine (4-AP) model. We injected the adeno-associated viral vector (AAV-PHP.eB-hSyn-KR2-YFP) containing the KR2 sodium pump gene enhanced with appropriate trafficking tags. KR2 expression was observed in the lateral entorhinal cortex and CA1 hippocampus. Using whole-cell patch clamp in mouse brain slices, we show that KR2, when stimulated with LED light, induces a substantial hyperpolarization of entorhinal neurons. However, continuous photostimulation of KR2 does not interrupt ictal discharges in mouse entorhinal cortex slices induced by a solution containing 4-AP. KR2-induced hyperpolarization strongly activates neuronal HCN channels. Consequently, turning off photostimulation resulted in HCN channel-mediated rebound depolarization accompanied by a transient increase in spontaneous network activity. Using low-frequency pulsed photostimulation, we induced the generation of short HCN channel-mediated discharges that occurred in response to the light stimulus being turned off; these discharges reliably interrupt ictal activity. Thus, low-frequency pulsed photostimulation of KR2 can be considered as a potential tool for controlling epileptic seizures.

Ferritin self-assembly, structure, function, and biotechnological applications.

Ferritin is a vital protein complex responsible for storing iron in almost all living organisms. It plays a crucial role in various metabolic pathways, inflammation processes, stress response, and pathogenesis of cancer and neurodegenerative diseases. In this review we discuss the role of ferritin in diseases, cellular iron regulation, its structural features, and its role in biotechnology. We also show that molecular mechanisms of ferritin self-assembly are key for a number of biotechnological and pharmaceutical applications. The assembly pathways strongly depend on the interface context of ferritin monomers and the stability of its different intermediate oligomers. To date, several schemes of self-assembly kinetics have been proposed. Here, we compare different self-assembly mechanisms and discuss the possibility of self-assembly control by switching between deadlock intermediate states.

ATP synthase FOF1 structure, function, and structure-based drug design.

ATP synthases are unique rotatory molecular machines that supply biochemical reactions with adenosine triphosphate (ATP)-the universal "currency", which cells use for synthesis of vital molecules and sustaining life. ATP synthases of F-type (FOF1) are found embedded in bacterial cellular membrane, in thylakoid membranes of chloroplasts, and in mitochondrial inner membranes in eukaryotes. The main functions of ATP synthases are control of the ATP synthesis and transmembrane potential. Although the key subunits of the enzyme remain highly conserved, subunit composition and structural organization of ATP synthases and their assemblies are significantly different. In addition, there are hypotheses that the enzyme might be involved in the formation of the mitochondrial permeability transition pore and play a role in regulation of the cell death processes. Dysfunctions of this enzyme lead to numerous severe disorders with high fatality levels. In our review, we focus on FOF1-structure-based approach towards development of new therapies by using FOF1 structural features inherited by the representatives of this enzyme family from different taxonomy groups. We analyzed and systematized the most relevant information about the structural organization of FOF1 to discuss how this approach might help in the development of new therapies targeting ATP synthases and design tools for cellular bioenergetics control.

Low-resolution structures of modular nanotransporters shed light on their functional activity.

Modular nanotransporters (MNTs) are multifunctional chimeric polypeptides for the multistep transport of locally acting cytotoxic agents into the nuclei of cancer target cells. MNTs consist of several polypeptide domains (functional modules) for the recognition of a cell-surface internalizable receptor, pH-dependent endosomal escape and subsequent transport into the nucleus through the nuclear pores. MNTs are a promising means for cancer treatment. As has been shown previously, all of the modules of MNTs retain their functionalities. Despite their importance, there is no structural information available about these chimeric polypeptides, which hampers the creation of new MNT variants. Here, a low-resolution 3D structure of an MNT is presented which was obtained by atomic force microscopy, transmission electron microscopy and small-angle X-ray scattering coupled to size-exclusion chromatography. The data suggest that the MNT can adopt two main conformations, but in both conformations the protein N- and C-termini are distanced and do not influence each other. The change in the MNT conformation during acidification of the medium was also studied. It was shown that the fraction of the elongated conformation increases upon acidification. The results of this work will be useful for the development of MNTs that are suitable for clinical trials and possible therapeutic applications.

A novel dimerization interface of cyclic nucleotide binding domain, which is disrupted in presence of cAMP: implications for CNG channels gating.

Cyclic nucleotide binding domain (CNBD) is a ubiquitous domain of effector proteins involved in signalling cascades of prokaryota and eukaryota. CNBD activation by cyclic nucleotide monophosphate (cNMP) is studied well in the case of several proteins. However, this knowledge is hardly applicable to cNMP-modulated cation channels. Despite the availability of CNBD crystal structures of bacterial cyclic nucleotide-gated (CNG) and mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels in presence and absence of the cNMP, the full understanding of CNBD conformational changes during activation is lacking. Here, we describe a novel CNBD dimerization interface found in crystal structures of bacterial CNG channel MlotiK1 and mammalian cAMP-activated guanine nucleotide-exchange factor Epac2. Molecular dynamics simulations show that the found interface is stable on the studied timescale of 100 ns, in contrast to the dimerization interface, reported previously. Comparisons with cN-bound structures of CNBD show that the dimerization is incompatible with cAMP binding. Thus, the cAMP-dependent monomerization of CNBD may be an alternative mechanism of the cAMP sensing. Based on these findings, we propose a model of the bacterial CNG channel modulation by cAMP.

X-ray-radiation-induced changes in bacteriorhodopsin structure.

Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates.