Temporal analysis of melanogenesis identifies fatty acid metabolism as key skin pigment regulator.

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating cutaneous hyperpigmentary conditions. Towards defining new potential targets, we followed temporal dynamics of melanogenesis using a cell-autonomous pigmentation model. Our study elucidates 3 dominant phases of synchronized metabolic and transcriptional reprogramming. The melanogenic trigger is associated with high MITF levels along with rapid uptake of glucose. The transition to pigmented state is accompanied by increased glucose channelisation to anabolic pathways that support melanosome biogenesis. SREBF1-mediated up-regulation of fatty acid synthesis results in a transient accumulation of lipid droplets and enhancement of fatty acids oxidation through mitochondrial respiration. While this heightened bioenergetic activity is important to sustain melanogenesis, it impairs mitochondria lately, shifting the metabolism towards glycolysis. This recovery phase is accompanied by activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism can resolve hyperpigmentary conditions in a guinea pig UV-tanning model. Our study reveals rewiring of the metabolic circuit during melanogenesis, and fatty acid metabolism as a potential therapeutic target in a variety of cutaneous diseases manifesting hyperpigmentary phenotype.

Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

Melanin protects skin cells from ultraviolet radiation-induced DNA damage. However, intermediates of eumelanin are highly reactive quinones that are potentially genotoxic. In this study, we systematically investigate the effect of sustained elevation of melanogenesis and map the consequent cellular repair response of melanocytes. Pigmentation increases γH2AX foci, DNA abasic sites, causes replication stress and invokes translesion polymerase Polκ in primary human melanocytes, as well as mouse melanoma cells. Confirming the causal link, CRISPR-based genetic ablation of tyrosinase results in depigmented cells with low Polκ levels. During pigmentation, Polκ activates replication stress response and keeps a check on uncontrolled proliferation of cells harboring melanin-damaged DNA. The mutational landscape observed in human melanoma could in part explain the error-prone bypass of DNA lesions by Polκ, whose absence would lead to genome instability. Thereby, translesion polymerase Polκ is a critical response of pigmenting melanocytes to combat melanin-induced DNA alterations. Our study illuminates the dark side of melanin and identifies (eu)melanogenesis as a key missing link between tanning response and mutagenesis, mediated via the necessary evil translesion polymerase, Polκ.

Kupyaphores are zinc homeostatic metallophores required for colonization of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host-pathogen interactions and disease outcome.

Histone variant dictates fate biasing of neural crest cells to melanocyte lineage.

In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro We demonstrate that this histone variant occupies nucleosomes in the promoter of the key melanocyte determinant mitf, and enhances its induction. CRISPR/Cas9-based targeted mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their regeneration. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches.

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygen-rich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis.

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca2+ levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca2+ stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.

pH-controlled histone acetylation amplifies melanocyte differentiation downstream of MITF.

Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. This involves the rapid and selective induction of melanocyte maturation genes, while concomitantly the expression of other effector genes is maintained. In this study, using cell-based and zebrafish model systems, we report on a pH-mediated feed-forward mechanism of epigenetic regulation that enables selective amplification of the melanocyte maturation program. We demonstrate that MITF activation directly elevates the expression of the enzyme carbonic anhydrase 14 (CA14). Nuclear localization of CA14 leads to an increase of the intracellular pH, resulting in the activation of the histone acetyl transferase p300/CBP. In turn, enhanced H3K27 histone acetylation at selected differentiation genes facilitates their amplified expression via MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in the formation of immature acidic melanocytes with decreased pigmentation, establishing a central role for this mechanism during melanocyte differentiation in vivo. Thus, we describe an epigenetic control system via pH modulation that reinforces cell fate determination by altering chromatin dynamics.

Myg1 exonuclease couples the nuclear and mitochondrial translational programs through RNA processing.

Semi-autonomous functioning of mitochondria in eukaryotic cell necessitates coordination with nucleus. Several RNA species fine-tune mitochondrial processes by synchronizing with the nuclear program, however the involved components remain enigmatic. In this study, we identify a widely conserved dually localized protein Myg1, and establish its role as a 3'-5' RNA exonuclease. We employ mouse melanoma cells, and knockout of the Myg1 ortholog in Saccharomyces cerevisiae with complementation using human Myg1 to decipher the conserved role of Myg1 in selective RNA processing. Localization of Myg1 to nucleolus and mitochondrial matrix was studied through imaging and confirmed by sub-cellular fractionation studies. We developed Silexoseqencing, a methodology to map the RNAse trail at single-nucleotide resolution, and identified in situ cleavage by Myg1 on specific transcripts in the two organelles. In nucleolus, Myg1 processes pre-ribosomal RNA involved in ribosome assembly and alters cytoplasmic translation. In mitochondrial matrix, Myg1 processes 3'-termini of the mito-ribosomal and messenger RNAs and controls translation of mitochondrial proteins. We provide a molecular link to the possible involvement of Myg1 in chronic depigmenting disorder vitiligo. Our study identifies a key component involved in regulating spatially segregated organellar RNA processing and establishes the evolutionarily conserved ribonuclease as a coordinator of nucleo-mitochondrial crosstalk.

Lesional skin in vitiligo exhibits delayed in vivo reepithelialization compared to the nonlesional skin.

Vitiligo, a common skin disorder, is characterized by the loss of functional melanocytes resulting in the depigmentation of skin. Previous studies have demonstrated molecular and architectural alterations in the epidermal keratinocytes upon loss of melanocytes. The physiological implications of these "altered" keratinocytes are yet not known. We investigated the wound healing efficiency of lesional vs nonlesional skin in 12 subjects with stable nonsegmental vitiligo using histological and ultrastructural evaluation of partial-thickness wounds. The wounds were examined 12 days postinjury, coinciding with the reepithelialization phase of healing marked primarily by keratinocyte migration and proliferation. This study demonstrated a significant difference in the reepithelialization potential between the lesional and nonlesional skin. While all 12 nonlesional wounds demonstrated considerable neoepidermis formation on the 12th day post wound, only four of the corresponding lesional samples showed comparable reepithelialization; the rest remaining in the inflammatory phase. Ultrastructural studies using transmission electron microscopy as well as immunohistochemical staining revealed a reduced number of desmosomes, shorter keratin tonofilaments and an increase in myofibroblast population in the dermis of lesional reepithelialized tissue compared to the nonlesional reepithelialized samples. This study implicates gross functional perturbations in the lesional skin during physiological wound healing in vitiligo, suggesting that the breakdown of keratinocyte-melanocyte network results in delayed wound repair kinetics in the lesional skin when compared to patient-matched nonlesional skin.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation.

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

Structural insights into the regulation of NADPH binding to reductase domains of nonribosomal peptide synthetases: A concerted loop movement model.

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4'-phosphopantetheine (4'-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis, where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes.

Multifaceted pathways protect human skin from UV radiation.

The recurrent interaction of skin with sunlight is an intrinsic constituent of human life, and exhibits both beneficial and detrimental effects. The apparent robust architectural framework of skin conceals remarkable mechanisms that operate at the interface between the surface and environment. In this Review, we discuss three distinct protective mechanisms and response pathways that safeguard skin from deleterious effects of ultraviolet (UV) radiation. The unique stratified epithelial architecture of human skin along with the antioxidant-response pathways constitutes the important defense mechanisms against UV radiation. The intricate pigmentary system and its intersection with the immune-system cytokine axis delicately balance tissue homeostasis. We discuss the relationship among these networks in the context of an unusual depigmenting disorder, vitiligo. The elaborate tunable mechanisms, elegant multilayered architecture and evolutionary selection pressures involved in skin and sunlight interaction makes this a compelling model to understand biological complexity.

Bioinspired Functionalized Melanin Nanovariants with a Range of Properties Provide Effective Color Matched Photoprotection in Skin.

Melanin and related polydopamine hold great promise; however, restricted fine-tunabilility limits their usefulness in biocompatible applications. In the present study, by taking a biomimetic approach, we synthesize peptide-derived melanin with a range of physicochemical properties. Characterization of these melanin polymers indicates that they exist as nanorange materials with distinct size distribution, shapes, and surface charges. These variants demonstrate similar absorption spectra but have different optical properties that correlate with particle size. Our approach enables incorporation of chemical groups to create functionalized polyvalent organic nanomaterials and enables customization of melanin. Further, we establish that these synthetic variants are efficiently taken up by the skin keratinocytes, display appreciable photoprotection with minimal cytotoxicity, and thereby function as effective color matched photoprotective agents. In effect we demonstrate that an array of functionalized melanins with distinct properties could be synthesized using bioinspired green chemistry, and these are of immense utility in generating customized melanin/polydopamine like materials.

The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes.

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys65, Arg68, and Arg69, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.

Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria.

Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order 'bi-bi' reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4'-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi-bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3-4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation-π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi-bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.

Identification and characterization of a type III polyketide synthase involved in quinolone alkaloid biosynthesis from Aegle marmelos Correa.

Quinolone alkaloids, found abundantly in the roots of bael (Aegle marmelos), possess various biological activities and have recently gained attention as potential lead molecules for novel drug designing. Here, we report the characterization of a novel Type III polyketide synthase, quinolone synthase (QNS), from A. marmelos that is involved in the biosynthesis of quinolone alkaloid. Using homology-based structural modeling, we identify two crucial amino acid residues (Ser-132 and Ala-133) at the putative QNS active site. Substitution of Ser-132 to Thr and Ala-133 to Ser apparently constricted the active site cavity resulting in production of naringenin chalcone from p-coumaroyl-CoA. Measurement of steady-state kinetic parameters demonstrates that the catalytic efficiency of QNS was severalfold higher for larger acyl-coenzymeA substrates as compared with smaller precursors. Our mutagenic studies suggest that this protein might have evolved from an evolutionarily related member of chalcone synthase superfamily by mere substitution of two active site residues. The identification and characterization of QNS offers a promising target for gene manipulation studies toward the production of novel alkaloid scaffolds.

Retrobiosynthetic approach delineates the biosynthetic pathway and the structure of the acyl chain of mycobacterial glycopeptidolipids.

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C(26)-C(34) fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.

Pigmented skin exhibits accelerated wound healing compared to the nonpigmented skin in Guinea pig model.

This study investigated and compared the wound healing kinetics of pigmented (PG) and non-pigmented (NP) skin in guinea pigs, focusing on histological and transcriptional changes. Full-thickness wounds created on PG and NP skin were evaluated at various time points post-injury. Fontana-Masson staining and ultrastructural analysis suggested the presence of melanin and melanosomes in PG skin, which coincided with an upregulation of melanogenic genes cKIT, TYR, and DCT. On day 9 post-wound, PG skin exhibited a rapid transition from the inflammatory to proliferative phase, which correlated with the reappearance of epidermal pigmentation whereas the NP skin exhibited a delayed neo-epidermis formation. Furthermore, the study revealed that melanocyte-derived growth factors (conditioned media) positively regulated keratinocyte migration while inhibiting fibroblast differentiation. These effects were more prominent in tyrosine-treated (hyperpigmented) melanocyte-CM as was TGF- ß expression. These findings provide valuable insights into the mechanisms underlying skin repair and pigmentation.

Synthesis, biological evaluation and docking studies of silicon incorporated diarylpyrroles as MmpL3 inhibitors: An effective strategy towards development of potent anti-tubercular agents.

Growing global demand for new molecules to treat tuberculosis has created an urgent need to develop novel strategies to combat the menace. BM212 related compounds were found to be potent anti-TB agents and they inhibit mycolic acid transporter, MmpL3, a known potent drug target from Mycobacterium tuberculosis. In order to enhance their inhibitory potency, several silicon analogues of diarylpyrroles related to BM212 were designed, synthesized, and evaluated for anti-tubercular activities. In Alamar blue assay, most of the silicon-incorporated compounds were found to be more potent than the parent compound (BM212), against Mycobacterium tuberculosis (MIC = 1.7 µM, H37Rv). Docking results from the crystal structure of MmpL3 and silicon analogues as pharmacophore model also strongly correlate with the biological assays and suggest that the incorporation of silicon in the inhibitor scaffold could enhance their potency by stabilizing the hydrophobic residues at the binding pocket. The best docking hit, compound 12 showed an MIC of 0.1 µM against H37Rv with an acceptable in vitro ADME profile and excellent selectivity index. Overall, the present study indicates that, the designed silicon analogues, especially compound 12 could be a good inhibitor for an intrinsically flexible drug-binding pocket of MmpL3 and has potential for further development as anti-tubercular agents.