A desert Chlorella sp. that thrives at extreme high-light intensities using a unique photoinhibition protection mechanism.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

Photocurrent Generation and Polarity Switching in Electrochemical Cells through Light-induced Excited State Proton Transfer of Photoacids and Photobases.

Light is a common source of energy in sustainable technologies for photocurrent generation. To date, in such light-harvesting applications, the excited electrons generate the photocurrent. Here, we introduce a new mechanism for photocurrent generation that is based on excited state proton transfer (ESPT) of photoacids and photobases that can donate or accept a proton, respectively, but only after excitation. We show that the formed ions following ESPT can either serve as electron donors or acceptors with the electrodes, or modify the kinetics of mass transport across the diffuse layer, both resulting in photocurrent generation. We further show that control of the current polarity is obtained by switching the irradiation between the photoacid and the photobase. Our study represents a new approach in photoelectrochemistry by introducing ESPT processes, which can be further utilized in light-responsive energy production or energy storage.

The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition.

Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities found that the alga employs all three known photoinhibition protection mechanisms: (i) massive reduction of the PSII antenna size; (ii) accumulation of protective carotenoids; and (iii) very rapid repair of photodamaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms, and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.

Variant PADI3 in Central Centrifugal Cicatricial Alopecia.

BACKGROUND: Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry. The disease is occasionally observed to affect women in families in a manner that suggests an autosomal dominant trait and usually manifests clinically after intense hair grooming. We sought to determine whether there exists a genetic basis of CCCA and, if so, what it is. METHODS: We used exome sequencing in a group of women with alopecia (discovery set), compared the results with those in a public repository, and applied other filtering criteria to identify candidate genes. We then performed direct sequencing to identify disease-associated DNA variations and RNA sequencing, protein modeling, immunofluorescence staining, immunoblotting, and an enzymatic assay to evaluate the consequences of potential etiologic mutations. We used a replication set that consisted of women with CCCA to confirm the data obtained with the discovery set. RESULTS: In the discovery set, which included 16 patients, we identified one splice site and three heterozygous missense mutations in PADI3 in 5 patients (31%). (The approximate prevalence of the disease is up to 5.6%.) PADI3 encodes peptidyl arginine deiminase, type III (PADI3), an enzyme that post-translationally modifies other proteins that are essential to hair-shaft formation. All three CCCA-associated missense mutations in PADI3 affect highly conserved residues and are predicted to be pathogenic; protein modeling suggests that they result in protein misfolding. These mutations were found to result in reduced PADI3 expression, abnormal intracellular localization of the protein, and decreased enzymatic activity - findings that support their pathogenicity. Immunofluorescence staining showed decreased expression of PADI3 in biopsy samples of scalp skin obtained from patients with CCCA. We then directly sequenced PADI3 in an additional 42 patients (replication set) and observed genetic variants in 9 of them. A post hoc analysis of the combined data sets showed that the prevalence of PADI3 mutation was higher among patients with CCCA than in a control cohort of women of African ancestry (P = 0.002 by the chi-square test; P = 0.006 by Fisher's exact test; and after adjustment for relatedness of persons, P = 0.03 and P = 0.04, respectively). CONCLUSIONS: Mutations in PADI3, which encodes a protein that is essential to proper hair-shaft formation, were associated with CCCA. (Funded by the Ram Family Foundation and others.).

Autosomal recessive congenital ichthyosis caused by a pathogenic missense variant in CLDN1.

Autosomal recessive congenital ichthyosis (ARCI) refers to a large and genetically heterogenous group of non-syndromic disorders of cornification featuring diffuse scaling. Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC) syndrome is a rare autosomal recessive syndromic form of ichthyosis. The disease usually results from premature termination codon-causing pathogenic variants in CLDN1 encoding CLAUDIN-1 (CLDN1). We used whole exome sequencing (WES), Sanger sequencing, 3D protein modeling, Western blotting, and immunofluorescence confocal microscopy to delineate the genetic basis of ichthyosis in two siblings with ichthyosis but no other ectodermal abnormalities. One of the two siblings underwent liver transplantation in early childhood due to biliary atresia. Both patients were found to carry a homozygous missense pathogenic variant, c.242G>A (p.Arg81His), in CLDN1. The variant resulted in decreased CLDN1 expression in patient skin. 3D protein modeling predicted that p.Arg81His induces deleterious conformational changes. Accordingly, HaCaT cells transfected with a construct expressing the mutant CLDN1 cDNA featured decreased levels and mislocation of CLDN1 as compared with cells expressing the wildtype cDNA. In conclusion, we describe the first pathogenic missense variant in CLDN1 shown to result in ARCI.

The Structural Basis for the Extraordinary Energy-Transfer Capabilities of the Phycobilisome.

Light absorption is the initial step in the photosynthetic process. In all species, most of the light is absorbed by dedicated pigment-protein complexes called light harvesting complexes or antenna complexes. In the case of cyanobacteria and red-algae, photosynthetic organisms found in a wide variety of ecological niches, the major antenna is called the Phycobilisome (PBS). The PBS has many unique characteristics that sets it apart from the antenna complexes of other organisms (bacteria, algae and plants). These differences include the type of light absorbing chromophores, the protein environment of the chromophores, the method of assembly and association and the intercellular location with respect to the photosynthetic reaction centers (RCs). Since the final goal of all antenna complexes is the same - controlled absorption and transfer of the energy of the sun to the RCs, the unique structural and chemical differences of the PBS also require unique energy transfer mechanisms and pathways. In this review we will describe in detail the structural facets that lead to a mature PBS, followed by an attempt to understand the energy transfer properties of the PBS as they have been measured experimentally.

Orange carotenoid protein burrows into the phycobilisome to provide photoprotection.

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5-10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.

Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences.

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis.

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway.

Structural heterogeneity leads to functional homogeneity in A. marina phycocyanin.

The major light harvesting antenna in all cyanobacterial species is the phycobilisome (PBS). The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single four-hexamer rod. We have determined the crystal structure of phycocyanin (AmPC), the major component of the A. marina PBS (AmPBS) to 2.1 Å. The basic unit of the AmPC is a heterodimer of two related subunits (α and ß), and we show that the asymmetric unit contains a superposition of two α and two ß isoforms, the products of the simultaneous expression of different genes. This is the first time to our knowledge that isolated proteins crystallized with such identifiable heterogeneity. We believe that the presence of the different isoforms allows the AmPBS to have a significant bathochromic shift in its fluorescence emission spectrum, allowing, in the total absence of allophycocyanin, a better overlap with absorption of the chlorophyll d-containing reaction centers. We show that this bathochromic shift exists in intact AmPBS as well as in its disassembled components, thus suggesting that AmPC can efficiently serve as the AmPBS terminal emitter.

Exome sequencing and network analysis identifies shared mechanisms underlying spinocerebellar ataxia.

The autosomal dominant cerebellar ataxias, referred to as spinocerebellar ataxias in genetic nomenclature, are a rare group of progressive neurodegenerative disorders characterized by loss of balance and coordination. Despite the identification of numerous disease genes, a substantial number of cases still remain without a genetic diagnosis. Here, we report five novel spinocerebellar ataxia genes, FAT2, PLD3, KIF26B, EP300, and FAT1, identified through a combination of exome sequencing in genetically undiagnosed families and targeted resequencing of exome candidates in a cohort of singletons. We validated almost all genes genetically, assessed damaging effects of the gene variants in cell models and further consolidated a role for several of these genes in the aetiology of spinocerebellar ataxia through network analysis. Our work links spinocerebellar ataxia to alterations in synaptic transmission and transcription regulation, and identifies these as the main shared mechanisms underlying the genetically diverse spinocerebellar ataxia types.

Concentration-based self-assembly of phycocyanin.

Cyanobacteria light-harvesting complexes can change their structure to cope with fluctuating environmental conditions. Studying in vivo structural changes is difficult owing to complexities imposed by the cellular environment. Mimicking this system in vitro is challenging, as well. The in vivo system is highly concentrated, and handling similar in vitro concentrated samples optically is difficult because of high absorption. In this research, we mapped the cyanobacteria antennas self-assembly pathways using highly concentrated solutions of phycocyanin (PC) that mimic the in vivo condition. PC was isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus and measured by several methods. PC has three oligomeric states: hexamer, trimer, and monomer. We showed that the oligomeric state was changed upon increase of PC solution concentration. This oligomerization mechanism may enable photosynthetic organisms to adapt their light-harvesting system to a wide range of environmental conditions.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

A crystal structure of 2-hydroxybiphenyl 3-monooxygenase with bound substrate provides insights into the enzymatic mechanism.

2-Hydroxybiphenyl 3-monooxygenase (HbpA) is an FAD dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. We have determined the structure of HbpA from the soil bacterium Pseudomonas azelaica HBP1 with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5Å to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. The entrance to the active site was confirmed by generating variant G255F which exhibited only 7% of the wild-type's specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications.

Cole Disease Results from Mutations in ENPP1.

The coexistence of abnormal keratinization and aberrant pigmentation in a number of cornification disorders has long suggested a mechanistic link between these two processes. Here, we deciphered the genetic basis of Cole disease, a rare autosomal-dominant genodermatosis featuring punctate keratoderma, patchy hypopigmentation, and uncommonly, cutaneous calcifications. Using a combination of exome and direct sequencing, we showed complete cosegregation of the disease phenotype with three heterozygous ENPP1 mutations in three unrelated families. All mutations were found to affect cysteine residues in the somatomedin-B-like 2 (SMB2) domain in the encoded protein, which has been implicated in insulin signaling. ENPP1 encodes ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which is responsible for the generation of inorganic pyrophosphate, a natural inhibitor of mineralization. Previously, biallelic mutations in ENPP1 were shown to underlie a number of recessive conditions characterized by ectopic calcification, thus providing evidence of profound phenotypic heterogeneity in ENPP1-associated genetic diseases.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

Investigation of phycobilisome subunit interaction interfaces by coupled cross-linking and mass spectrometry.

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.

Structural studies show energy transfer within stabilized phycobilisomes independent of the mode of rod-core assembly.

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2-5 cylinders surrounded by 6-8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.

Structure of the bovine COPI δ subunit µ homology domain at 2.15 Å resolution.

The heptameric COPI coat (coatomer) plays an essential role in vesicular transport in the early secretory system of eukaryotic cells. While the structures of some of the subunits have been determined, that of the δ-COP subunit has not been reported to date. The δ-COP subunit is part of a subcomplex with structural similarity to tetrameric clathrin adaptors (APs), where δ-COP is the structural homologue of the AP µ subunit. Here, the crystal structure of the µ homology domain (MHD) of δ-COP (δ-MHD) obtained by phasing using a combined SAD-MR method is presented at 2.15 Å resolution. The crystallographic asymmetric unit contains two monomers that exhibit short sections of disorder, which may allude to flexible regions of the protein. The δ-MHD is composed of two subdomains connected by unstructured linkers. Comparison between this structure and those of known MHD domains from the APs shows significant differences in the positions of specific loops and ß-sheets, as well as a more general change in the relative positions of the protein subdomains. The identified difference may be the major source of cargo-binding specificity. Finally, the crystal structure is used to analyze the potential effect of the I422T mutation in δ-COP previously reported to cause a neurodegenerative phenotype in mice.