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.

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.

Retinal-Based Anion Pump from the Cyanobacterium Tolypothrix campylonemoides.

In this work, TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was characterized. Analysis of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid from the TSA motif of the known halorhodopsin chloride pump. The TcaR protein was expressed in E. coli, purified, and incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one side of the membrane surface, as well as by fluorescence using the voltage-dependent dye oxonol VI. We have shown that TcaR rhodopsin functions as a powerful anion pump. Our results show that the novel microbial anion transporter, TcaR, deserves deeper investigation and may be of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.

Rhodopsin Channel Activity Can Be Evaluated by Measuring the Photocurrent Voltage Dependence in Planar Bilayer Lipid Membranes.

The studies of the functional properties of retinal-containing proteins often include experiments in model membrane systems, e.g., measurements of electric current through planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one of the membrane surfaces. However, the possibilities of this method have not been fully explored yet. We demonstrated that the voltage dependence of stationary photocurrents for two light-sensitive proteins, bacteriorhodopsin (bR) and channelrhodopsin 2 (ChR2), in the presence of protonophore had very different characteristics. In the case of the bR (proton pump), the photocurrent through the BLM did not change direction when the polarity of the applied voltage was switched. In the case of the photosensitive channel protein ChR2, the photocurrent increased with the increase in voltage and the current polarity changed with the change in the voltage polarity. The protonophore 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB) was more efficient in the maximizing stationary photocurrents. In the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), the amplitude of the measured photocurrents for bR significantly decreased, while in the case of ChR2, the photocurrents virtually disappeared. The difference between the effects of TTFB and CCCP was apparently due to the fact that, in contrast to TTFB, CCCP transfers protons across the liposome membranes with a higher rate than through the decane-containing BLM used as a surface for the proteoliposome adsorption.

On the Role of Normal Aging Processes in the Onset and Pathogenesis of Diseases Associated with the Abnormal Accumulation of Protein Aggregates.

Aging is a prime systemic cause of various age-related diseases, in particular, proteinopathies. In fact, most diseases associated with protein misfolding are sporadic, and their incidence increases with aging. This review examines the process of protein aggregate formation, the toxicity of such aggregates, the organization of cellular systems involved in proteostasis, and the impact of protein aggregates on important cellular processes leading to proteinopathies. We also analyze how manifestations of aging (mitochondrial dysfunction, dysfunction of signaling systems, changes in the genome and epigenome) facilitate pathogenesis of various proteinopathies either directly, by increasing the propensity of key proteins for aggregation, or indirectly, through dysregulation of stress responses. Such analysis might help in outlining approaches for treating proteinopathies and extending healthy longevity.

Highly Sensitive Coherent Anti-Stokes Raman Scattering Imaging of Protein Crystals.

Serial crystallography at last generation X-ray synchrotron sources and free electron lasers enabled data collection with micrometer and even submicrometer size crystals, which have resulted in amazing progress in structural biology. However, imaging of small crystals, which although is highly demanded, remains a challenge, especially in the case of membrane protein crystals. Here we describe a new extremely sensitive method of the imaging of protein crystals that is based on coherent anti-Stokes Raman scattering.

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.

Order wrapped in chaos: On the roles of intrinsically disordered proteins and RNAs in the arrangement of the mitochondrial enzymatic machines.

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and non-coding RNAs in mitochondrial organization.

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.

Mirror proteorhodopsins.

Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name "mirror proteorhodopsins", from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer.

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.

Crystal structure of semi-synthetic obelin-v after calcium induced bioluminescence implies coelenteramine as the main reaction product.

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.

Mechanisms of membrane protein crystallization in 'bicelles'.

Despite remarkable progress, mainly due to the development of LCP and 'bicelle' crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the "bicelle" crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of "bicelle" MP crystallization and will support rational design of crystallization.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review.

Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.

Role of the HAMP domain region of sensory rhodopsin transducers in signal transduction.

Archaea are able to sense light via the complexes of sensory rhodopsins I and II and their corresponding chemoreceptor-like transducers HtrI and HtrII. Though generation of the signal has been studied in detail, the mechanism of its propagation to the cytoplasm remains obscured. The cytoplasmic part of the transducer consists of adaptation and kinase activity modulating regions, connected to transmembrane helices via two HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, phosphatases) domains. The inter-HAMP region of Natronomonas pharaonis HtrII (NpHtrII) was found to be α-helical [Hayashi, K., et al. (2007) Biochemistry 46, 14380-14390]. We studied the inter-HAMP regions of NpHtrII and other phototactic signal transducers by means of molecular dynamics. Their structure is found to be a bistable asymmetric coiled coil, in which the protomers are longitudinally shifted by ~1.3 Å. The free energy penalty for the symmetric structure is estimated to be 1.2-1.5 kcal/mol depending on the molarity of the solvent. Both flanking HAMP domains are mechanistically coupled to the inter-HAMP region and are asymmetric. The longitudinal shift in the inter-HAMP region is coupled with the in-plane displacement of the cytoplasmic part by 8.6 Å relative to the transmembrane part. The established properties suggest that (1) the signal may be transduced through the inter-HAMP domain switching and (2) the inter-HAMP region may allow cytoplasmic parts of the transducers to come sufficiently close to each other to form oligomers.

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II-transducer complex.

Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII-NpHtrII) was performed. The influence of the polydispersity of NpSRII-NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

All-d-Enantiomeric Peptide D3 Designed for Alzheimer's Disease Treatment Dynamically Interacts with Membrane-Bound Amyloid-ß Precursors.

Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-ß peptide (Aß) oligomers play a crucial role in AD pathogenesis. All-d-Enantiomeric peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aß aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aß monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aß sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aß conversion into ß-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.

Molecular model of a sensor of two-component signaling system.

Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein.

Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During Aspergillus fumigatus-Induced Inflammation.

People are constantly exposed to airborne fungal spores, including Aspergillus fumigatus conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense A. fumigatus conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the A. fumigatus conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII+ antigen-presenting cells, including IE-DCs, and CD11b+ phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of A. fumigatus conidia increased the number of CD11b+ phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b+ phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b+ phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b+ phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b+ phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.