Mitophagy mediated by BNIP3 and NIX protects against ferroptosis by downregulating mitochondrial reactive oxygen species.

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

Chromophore-Protein Interactions Affecting the Polyene Twist and π-π* Energy Gap of the Retinal Chromophore in Schizorhodopsins.

The properties of a prosthetic group are broadened by interactions with its neighboring residues in proteins. The retinal chromophore in rhodopsins absorbs light, undergoes structural changes, and drives functionally important structural changes in proteins during the photocycle. It is therefore crucial to understand how chromophore-protein interactions regulate the molecular structure and electronic state of chromophores in rhodopsins. Schizorhodopsin is a newly discovered subfamily of rhodopsins found in the genomes of Asgard archaea, which are extant prokaryotes closest to the last common ancestor of eukaryotes and of other microbial species. Here, we report the effects of a hydrogen bond between a retinal Schiff base and its counterion on the twist of the polyene chain and the color of the retinal chromophore. Correlations between spectral features revealed the unexpected fact that the twist of the polyene chain is reduced as the hydrogen bond becomes stronger, suggesting that the twist is caused by tight atomic contacts between the chromophore and nearby residues. In addition, the strength of the hydrogen bond is the primary factor affecting the color-tuning of the retinal chromophore in schizorhodopsins. The findings of this study are valuable for manipulating the molecular structure and electronic state of the chromophore by controlling chromophore-protein interactions.

Cis-Trans Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping.

The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward proton-pumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis-trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis-trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.

Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin.

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr-Ala-Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

Functional characterization of four opsins and two G alpha subtypes co-expressed in the molluscan rhabdomeric photoreceptor.

BACKGROUND: Rhabdomeric photoreceptors of eyes in the terrestrial slug Limax are the typical invertebrate-type but unique in that three visual opsins (Gq-coupled rhodopsin, xenopsin, Opn5A) and one retinochrome, all belonging to different groups, are co-expressed. However, molecular properties including spectral sensitivity and G protein selectivity of any of them are not determined, which prevents us from understanding an advantage of multiplicity of opsin properties in a single rhabdomeric photoreceptor. To gain insight into the functional role of the co-expression of multiple opsin species in a photoreceptor, we investigated the molecular properties of the visual opsins in the present study. RESULTS: First, we found that the fourth member of visual opsins, Opn5B, is also co-expressed in the rhabdomere of the photoreceptor together with previously identified three opsins. The photoreceptors were also demonstrated to express Gq and Go alpha subunits. We then determined the spectral sensitivity of the four visual opsins using biochemical and spectroscopic methods. Gq-coupled rhodopsin and xenopsin exhibit maximum sensitivity at ~ 456 and 475 nm, respectively, and Opn5A and Opn5B exhibit maximum sensitivity at ~ 500 and 470 nm, respectively, with significant UV sensitivity. Notably, in vitro experiments revealed that Go alpha was activated by all four visual opsins, in contrast to the specific activation of Gq alpha by Gq-coupled rhodopsin, suggesting that the eye photoreceptor of Limax uses complex G protein signaling pathways. CONCLUSIONS: The eye photoreceptor in Limax expresses as many as four different visual opsin species belonging to three distinct classes. The combination of opsins with different spectral sensitivities and G protein selectivities may underlie physiological properties of the ocular photoreception, such as a shift in spectral sensitivity between dark- and light-adapted states. This may be allowed by adjustment of the relative contribution of the four opsins without neural networks, enabling a simple strategy for fine-tuning of vision.

Application of linear and machine learning models to genomic prediction of fatty acid composition in Japanese Black cattle.

We collected 3180 records of oleic acid (C18:1) and monounsaturated fatty acid (MUFA) measured using gas chromatography (GC) and 6960 records of C18:1 and MUFA measured using near-infrared spectroscopy (NIRS) in intermuscular fat samples of Japanese Black cattle. We compared genomic prediction performance for four linear models (genomic best linear unbiased prediction [GBLUP], kinship-adjusted multiple loci [KAML], BayesC, and BayesLASSO) and five machine learning models (Gaussian kernel [GK], deep kernel [DK], random forest [RF], extreme gradient boost [XGB], and convolutional neural network [CNN]). For GC-based C18:1 and MUFA, KAML showed the highest accuracies, followed by BayesC, XGB, DK, GK, and BayesLASSO, with more than 6% gain of accuracy by KAML over GBLUP. Meanwhile, DK had the highest prediction accuracy for NIRS-based C18:1 and MUFA, but the difference in accuracies between DK and KAML was slight. For all traits, accuracies of RF and CNN were lower than those of GBLUP. The KAML extends GBLUP methods, of which marker effects are weighted, and involves only additive genetic effects; whereas machine learning methods capture non-additive genetic effects. Thus, KAML is the most suitable method for breeding of fatty acid composition in Japanese Black cattle.

Photochemistry of the Retinal Chromophore in Microbial Rhodopsins.

Microbial rhodopsins are photoreceptive membrane proteins of microorganisms that express diverse photobiological functions. All-trans-retinylidene Schiff base, the so-called all-trans-retinal, is a chromophore of microbial rhodopsins, which captures photons. It isomerizes into the 13-cis form upon photoexcitation. Isomerization of retinal leads to sequential conformational changes in the protein, giving rise to active states that exhibit biological functions. Despite the rapidly expanding diversity of microbial rhodopsin functions, the photochemical behaviors of retinal were considered to be common among them. However, the retinal of many recently discovered rhodopsins was found to exhibit new photochemical characteristics, such as highly red-shifted absorption, isomerization to 7-cis and 11-cis forms, and energy transfer from a secondary carotenoid chromophore to the retinal, which is markedly different from that established in canonical microbial rhodopsins. Here, I review new aspects of retinal found in novel microbial rhodopsins and highlight the emerging problems that need to be addressed to understand noncanonical retinal photochemistry.

Effects of the Unique Chromophore-Protein Interactions on the Primary Photoreaction of Schizorhodopsin.

Schizorhodopsin (SzR) is a newly discovered microbial rhodopsin subfamily, functioning as an unusual inward-proton (H+) pump upon absorbing light. Two major protein structural differences around the chromophore have been found, resulting in unique chromophore-protein interactions that may be responsible for its unusual function. Therefore, it is important to elucidate how such a difference affects the primary photoreaction dynamics. We study the primary dynamics of SzR and its C75S mutant by femtosecond time-resolved absorption (TA) spectroscopy. The obtained TA data revealed that the photoisomerization in SzR proceeds more slowly and less efficiently than typical outward H+-pumping rhodopsins and that it further slows in the C75S mutant. We performed impulsive stimulated Raman measurements to clarify the effect of the cysteine residue on the retinal chromophore and found that interactions with Cys75 flatten the retinal chromophore of wild-type SzR. We discuss the effect of the unique chromophore-cysteine interaction on the retinal isomerization dynamics and structure of SzR.

Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes.

Ion pumps are membrane proteins that actively translocate ions by using energy. All known pumps bind ions in the resting state, and external energy allows ion transport through protein structural changes. The light-driven sodium-ion pump Krokinobacter eikastus rhodopsin 2 (KR2) is an exceptional case in which ion binding follows the energy input. In this study, we report another case of this unusual transport mode. The NTQ rhodopsin from Alteribacter aurantiacus (AaClR) is a natural light-driven chloride pump, in which the chloride ion binds to the resting state. AaClR is also able to pump sulfate ions, though the pump efficiency is much lower for sulfate ions than for chloride ions. Detailed spectroscopic analysis revealed no binding of the sulfate ion to the resting state of AaClR, indicating that binding of the substrate (sulfate ion) to the resting state is not necessary for active transport. This property of the AaClR sulfate pump is similar to that of the KR2 sodium pump. Photocycle dynamics of the AaClR sulfate pump resemble a non-functional cycle in the absence of anions. Despite this, flash photolysis and difference Fourier transform infrared spectroscopy suggest transient binding of the sulfate ion to AaClR. The molecular mechanism of this unusual active transport by AaClR is discussed.

Evaluating the performance of genomic prediction accounting for effects of single nucleotide polymorphism markers in reproductive traits of Japanese Black cattle.

We examined the prediction accuracies of genomic best linear unbiased prediction (GBLUP), various weighted GBLUP according to the degrees of marker effects (WGBLUP) and machine learning (ML) methods, and compared them with traditional BLUP for age at first calving (AFC), calving difficulty (CD), and gestation length in Japanese Black cattle. For WGBLUP, firstly, BayesC and FarmCPU were used to estimate marker effects. Then, we constructed three weighted genomic relationship matrices from information of estimated marker effects in the first step: absolute value of the estimated marker-effect WGBLUP, estimated marker-variance WGBLUP, and genomic-feature WGBLUP. For ML, we applied Gaussian kernel, random forest, extreme gradient boost, and support vector regression. We collected a total of 2583 animals having both phenotypic records and genotypes with 30,105 markers and 16,406 pedigree records. For AFC, prediction accuracies of WGBLUP methods using FarmCPU exceeded BLUP by 25.7%-39.5%. For CD, estimated marker-variance WGBLUP using BayesC achieved the highest prediction accuracy. Among ML methods, extreme gradient boost, support vector regression, and Gaussian kernel increased prediction accuracies by 28.4%, 19.0%, and 36.4% for AFC, CD, and gestation length compared with BLUP, respectively. Thus, prediction performance could be improved using suitable WGBLUP and ML methods according to target reproductive traits for the population used.

Comparing pedigree and genomic inbreeding coefficients, and inbreeding depression of reproductive traits in Japanese Black cattle.

BACKGROUND: Pedigree-based inbreeding coefficients have been generally included in statistical models for genetic evaluation of Japanese Black cattle. The use of genomic data is expected to provide precise assessment of inbreeding level and depression. Recently, many measures have been used for genome-based inbreeding coefficients; however, with no consensus on which is the most appropriate. Therefore, we compared the pedigree- ([Formula: see text]) and multiple genome-based inbreeding coefficients, which were calculated from the genomic relationship matrix with observed allele frequencies ([Formula: see text]), correlation between uniting gametes ([Formula: see text]), the observed vs expected number of homozygous genotypes ([Formula: see text]), runs of homozygosity (ROH) segments ([Formula: see text]) and heterozygosity by descent segments ([Formula: see text]). We quantified inbreeding depression from estimating regression coefficients of inbreeding coefficients on three reproductive traits: age at first calving (AFC), calving difficulty (CD) and gestation length (GL) in Japanese Black cattle. RESULTS: The highest correlations with [Formula: see text] were for [Formula: see text] (0.86) and [Formula: see text] (0.85) whereas [Formula: see text] and [Formula: see text] provided weak correlations with [Formula: see text], with range 0.33-0.55. Except for [Formula: see text] and [Formula: see text], there were strong correlations among genome-based inbreeding coefficients ([Formula: see text] 0.94). The estimates of regression coefficients of inbreeding depression for [Formula: see text] was 2.1 for AFC, 0.63 for CD and -1.21 for GL, respectively, but [Formula: see text] had no significant effects on all traits. Genome-based inbreeding coefficients provided larger effects on all reproductive traits than [Formula: see text]. In particular, for CD, all estimated regression coefficients for genome-based inbreeding coefficients were significant, and for GL, that for [Formula: see text] had a significant.. Although there were no significant effects when using overall genome-level inbreeding coefficients for AFC and GL, [Formula: see text] provided significant effects at chromosomal level in four chromosomes for AFC, three chromosomes for CD, and two chromosomes for GL. In addition, similar results were obtained for [Formula: see text]. CONCLUSIONS: Genome-based inbreeding coefficients can capture more phenotypic variation than [Formula: see text]. In particular, [Formula: see text] and [Formula: see text] can be considered good estimators for quantifying inbreeding level and identifying inbreeding depression at the chromosome level. These findings might improve the quantification of inbreeding and breeding programs using genome-based inbreeding coefficients.

Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.

A light-gated cation channel with high reactivity to weak light.

The cryptophyte algae, Guillardia theta, possesses 46 genes that are homologous to microbial rhodopsins. Five of them are functionally light-gated cation channelrhodopsins (GtCCR1-5) that are phylogenetically distinct from chlorophyte channelrhodopsins (ChRs) such as ChR2 from Chlamydomonas reinhardtii. In this study, we report the ion channel properties of these five CCRs and compared them with ChR2 and other ChRs widely used in optogenetics. We revealed that light sensitivity varied among GtCCR1-5, in which GtCCR1-3 exhibited an apparent EC50 of 0.21-1.16 mW/mm2, similar to that of ChR2, whereas GtCCR4 and GtCCR5 possess two EC50s, one of which is significantly small (0.025 and 0.032 mW/mm2). GtCCR4 is able to trigger action potentials in high temporal resolution, similar to ChR2, but requires lower light power, when expressed in cortical neurons. Moreover, a high light-sensitive response was observed when GtCCR4 was introduced into blind retina ganglion cells of rd1, a mouse model of retinitis pigmentosa. Thus, GtCCR4 provides optogenetic neuronal activation with high light sensitivity and temporal precision.

The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy.

Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.

Time-resolved detection of light-induced conformational changes of heliorhodopsin.

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 µs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 µs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.

Hva22, a REEP family protein in fission yeast, promotes reticulophagy in collaboration with a receptor protein.

The endoplasmic reticulum (ER) undergoes selective autophagy called reticulophagy or ER-phagy. Multiple reticulon- and receptor expression enhancing protein (REEP)-like ER-shaping proteins, including budding yeast Atg40, serve as reticulophagy receptors that stabilize the phagophore on the ER by interacting with phagophore-conjugated Atg8. Additionally, they facilitate phagophore engulfment of the ER by remodeling ER morphology. We reveal that Hva22, a REEP family protein in fission yeast, promotes reticulophagy without Atg8-binding capacity. The role of Hva22 in reticulophagy can be replaced by expressing Atg40 independently of its Atg8-binding ability. Conversely, adding an Atg8-binding sequence to Hva22 enables it to substitute for Atg40 in budding yeast. Thus, the phagophore-stabilizing and ER-shaping activities, both of which Atg40 solely contains, are divided between two separate factors, receptors and Hva22, respectively, in fission yeast.Abbreviations: AIM: Atg8-family interacting motif; Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum GFP: green fluorescent protein; NAA: 1-naphthaleneacetic acid; REEP: receptor expression enhancing protein; RFP: red fluorescent protein; UPR: unfolded protein response.

VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals.

Vascular endothelial growth factor A (VEGF-A) plays pivotal roles in regulating tumor angiogenesis as well as physiological vascular function. The major VEGF-A isoforms, VEGF-A121 and VEGF-A165, in serum, plasma, and platelets have not been exactly evaluated due to the lack of the appropriate assay system. Antibodies against human VEGF-A121 and VEGF-A165 (hVEGF-A121 and hVEGF-A165) were successfully produced and Enzyme-Linked ImmunoSorbent Assay (ELISA) for hVEGF-A121 and hVEGF-A165 were separately created by these monoclonal antibodies. The measurement of recombinant hVEGF-A121 and hVEGF-A165 by the created ELISA showed no cross-reaction between hVEGF-A121 and hVEGF-A165 in conditioned media from HEK293 cells transfected with either hVEGF-A121 or hVEGF-A165 expression vector. The levels of VEGF-A121 and VEGF-A165 in serum, plasma, and platelets from 59 healthy volunteers proved that VEGF-A121 level was higher than VEGF-A165 in both plasma and serum in all the cases. VEGF-A121 or VEGF-A165 in serum represented higher level than that in plasma. In contrast, the level of VEGF-A165 was higher than VEGF-A121 in platelets. The newly developed ELISAs for hVEGF-A121 and hVEGF-A165 revealed different ratios of VEGF isoforms in serum, plasma, and platelets. Measuring these isoforms in combination provides useful information as biomarkers for diseases involving VEGF-A121 and VEGF-A165.

Converting a Natural-Light-Driven Outward Proton Pump Rhodopsin into an Artificial Inward Proton Pump.

Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.