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Results for “Biochemistry”

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bioRxiv2017

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term early risers. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

biochemistry↗

bioRxiv2017

ETV4 And AP1 Transcription Factors Form Multivalent Interactions With Three Sites On The MED25 Activator-Interacting Domain

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. Within the ETS family, high-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker or no detectable binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-binding sequences were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

biochemistry↗

bioRxiv2017

Chaperonin facilitates protein folding by avoiding polypeptide collapse

Chaperonins assist folding of many cellular proteins, including essential proteins for cell viability. However, it remains unclear how chaperonin-assisted folding is different from spontaneous folding. Chaperonin GroEL/GroES facilitates folding of denatured protein encapsulated in its central cage but the denatured protein often escapes from the cage to the outside during reaction. Here, we show evidence that the in-cage-folding and the escape occur diverging from the same intermediate complex in which polypeptide is tethered loosely to the cage and partly protrudes out of the cage. Furthermore, denatured proteins in the chaperonin cage start their folding from extended conformations but not from compact conformations as usually observed in spontaneous folding. We propose that the formation of tethered intermediate of polypeptide is necessary to prevent polypeptide collapse at the expense of polypeptide escape. The tethering of polypeptide would allow freely mobile portions of tethered polypeptide to fold segmentally. The folding acceleration and deceleration by chaperonin for various substrate proteins can be explained by considering the tethering.

biochemistry↗

bioRxiv2017

Environmental cystine drives glutamine anaplerosis and sensitizes cells to glutaminase inhibition

Many cancer cell lines depend on extracellular glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation in vitro. However, recent studies have suggested that some cells that depend on glutamine anaplerosis in culture rely much less on glutamine catabolism to proliferate in vivo, with environmental differences between tumors and cell culture influencing the extent of glutamine catabolism. Here we sought to better understand the environmental differences that cause differential dependence on glutamine for TCA cycle anaplerosis. We find that cells cultured in adult bovine serum, a condition that more closely reflects the nutrients available to cells in vivo, leads to decreased glutamine catabolism and reliance on glutamine anaplerosis compared to standard tissue culture conditions. By analyzing the nutrient differences between bovine serum and media, we find that levels of a single nutrient, cystine, can account for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11, and that environmental cystine levels in conjunction with xCT/SLC7A11 expression is necessary and sufficient to drive increased glutamine anaplerosis, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer cells.

biochemistry↗

bioRxiv2017

A commensal adhesin enhances E. coli retention by mucin, while mucin desulfation by mucin-foraging bacteria enhances its transmigration through the mucus barrier

Bacterial adhesion to host receptors is an early and essential step in bacterial colonization, and the nature of adhesion-receptor interactions determines bacterial localization and thus the outcome of these interactions. Here, we determine the host receptors for the Multivalent Adhesion Molecule from the gut commensal E. coli HS (MAMHS), which contains an array of seven mammalian cell entry (MCE) domains. The MAMHS adhesin interacts with a range of host receptors, through recognition of a shared 3-O-sulfo-galactosyl moiety. This functional group is also found in mucin, a component of the intestinal mucus layer and thus one of the prime adherence targets for commensal E. coli. Mucin gels impede the motility of E. coli by acting as a physical barrier, and the barrier effect is enhanced by specific interactions between mucin and MAMHS in a sulfation-dependent manner. Desulfation of mucin by pure sulfatase or the sulfatase-producing commensal Bacteroides thetaiotaomicron decreases binding of E. coli to mucin and increases attachment of bacteria to the epithelial surface, through interactions with surface-localized sulfated lipid and protein receptors. Together, our results demonstrate that the E. coli adhesin MAMHS facilitates retention of a gut commensal by mucin, and suggest that the amount of sulfatase secreted by mucin-foraging bacteria inhabiting the same niche, such as B. thetaiotaomicron, may affect the capacity of the mucus barrier to retain commensal E. coli.

biochemistry↗

bioRxiv2017

Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions including metabolic (glycolytic or mitochondrial) inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations, indicating a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.

biochemistry↗

bioRxiv2017

Binding Of A Newly Identified Essential Light Chain To Expressed Plasmodium falciparum Class XIV Myosin Enhances Actin Motility

Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional \"essential\" light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 m/sec, more than twice that of PfMyoA-MTIP (~1.7 m/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.\n\nSignificanceMotility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, relies on a divergent actomyosin system powered by the class XIV myosin, PfMyoA. We show that functional PfMyoA can be expressed in Sf9 cells if a Plasmodium spp. myosin chaperone is co-expressed. We identified an \"essential\" light chain (PfELC) that binds to PfMyoA in parasites. In vitro expression of PfMyoA heavy chain with PfELC and the known light chain MTIP produced the fastest speeds of actin movement (~3.8 m/sec). Duty ratio estimates suggest that ~6 PfMyoA motors can move actin at maximal speed, a feature that may facilitate interaction with short, dynamic Plasmodium actin filaments. Our findings enable drug screening for myosin-based inhibitors of Plasmodium cellular invasion.

biochemistry↗

bioRxiv2017

TGH: Trans-Generational Hormesis And The Inheritance Of Aging Resistance

Animals respond to dietary changes by adapting their metabolism to available nutrients through insulin and insulin-like growth factor signalling. Restricting calorie intake generally extends life and health span, but Drosophila fed non-ideal sugars such as galactose are stressed and have shorter life spans. Here, we report that although these flies have shorter life spans, their offspring show significant life extension if switched to a normal sugar (glucose) diet. We define this as TGH or trans-generational hormesis, a beneficial effect that comes from a mild stress. We trace the effects to changes in stress responses in parents, ROS production, effects on lipid metabolism, and changes in chromatin and gene expression. We find that this mechanism is similar to what happens to the long lived Indy mutants on normal food, but surprisingly find that Indy is required for life span extension for galactose fed flies. Indy mutant flies grown on galactose do not live longer as do their siblings grown on glucose, rather overexpression of Indy rescues lifespan for galactose reared flies. We define a process where sugar metabolism can generate epigenetic changes that are inherited by offspring, providing a mechanism for how transgenerational nutrient sensitivities are passed on.

biochemistry↗

bioRxiv2017

Glutathione And Thioredoxin Systems Of The Malaria Parasite Plasmodium falciparum: Partners In Crime?

In P. falciparum, antioxidant proteins of the glutathione and thioredoxin systems are compartmentalized. Some subcellular compartments have only a partial complement of these proteins. This lack of key anti-oxidant proteins in certain sub-cellular compartments might be compensated by functional complementation between these systems. By assessing the cross-talk between these systems, we show for the first time, that the glutathione system can reduce thioredoxins that are poor substrates for thioredoxin reductase (Thioredoxin-like protein 1 and Thioredoxin 2) and thioredoxins that lack access to thioredoxin reductase (Thioredoxin 2). Our data suggests that crosstalk between the glutathione and thioredoxin systems does exist; this could compensate for the absence of certain antioxidant proteins from key subcellular compartments.

biochemistry↗

bioRxiv2017

Ubiquitin and ATP-dependent unfoldase activity of P97/VCP·NPLOC4·UFD1L1 is enhanced by a mutation that causes multisystem proteinopathy

p97 is a segregase that plays a key role in numerous ubiquitin-dependent pathways, such as ER-associated degradation (ERAD). It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate--Ub-GFP modified with K48-linked ubiquitin chains--for in vitro p97 activity assays. We demonstrate for the first time that wild type p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Pagets Disease of bone, and frontotemporal dementia (IBMPFD) in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

biochemistry↗

bioRxiv2017

Disabling Cas9 by an anti-CRISPR DNA mimic

CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 [A] resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

biochemistry↗

bioRxiv2017

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

biochemistry↗

bioRxiv2017

Chimeric 14-3-3 Proteins For Unravelling Interactions With Intrinsically Disordered Partners

In eukaryotes, several proteins act as \"hubs\", integrating signals from a variety of interacting partners that bind to the hub through intrinsically disordered regions. Not surprisingly, one of the major hubs, the 14-3-3 protein, that plays wide-ranging roles in cellular processes, has been linked with a number of disorders including neurodegenerative diseases and cancer. A partner protein usually binds with its phosphopeptide accommodated in an amphipathic groove (AG) of 14-3-3, a promising platform for therapeutic intervention. Protein plasticity in the groove allows to accommodate a range of phosphopeptides with different sequences. So far, in spite of mammoth effort, accurate structural information has been derived only for few 14-3-3 complexes with phosphopeptide-containing proteins or various short synthetic peptides. The progress has been prevented by intrinsic disorder of partner proteins and, in case of transient interactions, by the low affinity of phosphopeptides. We reasoned that these problems could be resolved by using chimeric 14-3-3 proteins with incorporated peptide sequences. We tested this hypothesis and found that such chimeric proteins are easy to design, express, purify and crystallize. We show that when attached to the C terminus of 14-3-3 via an optimal linker, peptides become stoichiometrically phosphorylated by protein kinase A during bacterial co-expression. We determined crystal structures for complexes of chimeric 14-3-3 protein fused with three different peptides. In most of the cases, the phosphopeptide is bound inside the AG, providing invaluable information on its interaction with the protein. This approach can reinvigorate studies of 14-3-3 protein complexes, including those with otherwise challenging low affinity phosphopeptides. Furthermore, 14-3-3-phosphopeptide chimeras can be useful for the design of novel biosensors for in vitro and in vivo imaging experiments.

biochemistry↗

bioRxiv2017

The Yeast Mig1 Transcriptional Repressor Is Dephosphorylated By Glucose-Dependent And Independent Mechanisms

Saccharomyces cerevisiae AMPK/Snf1 regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

biochemistry↗

bioRxiv2017

A Dry Method For Preserving Tear Samples

Tears covering the ocular surface is an important bio-fluid containing thousands of molecules, including proteins, lipids, metabolites, nucleic acids, and electrolytes. Tears are valuable resources for biomarker research of ocular and even systemic diseases. For application in biomarker studies, tear samples should ideally be stored using a simple, low-cost, and efficient method along with the patients medical records. For this purpose, we developed a novel Schirmers strip-based dry method that allows for storage of tear samples in vacuum bags at room temperature. Using this method, tear protein patterns can also be preserved. Liquid chromatography-mass spectrometry/mass spectrometry analysis of proteins recovered from the dry method and traditional wet method showed no significant difference. Some tissue/organ enriched proteins were identified in tear, thus tear might be a good window for monitoring the change of these tissues or organs. This dry method facilitates sample transportation and enables the storage of tear samples on a large scale, increasing the availability of samples for studying disease biomarkers in tears.

biochemistry↗

bioRxiv2017

Alternative cleavage of a bone morphogenetic protein (BMP) produces ligands with distinct developmental functions and receptor preference

TGF-{beta} and Bone Morphogenetic Protein (BMP) family proteins are made as proprotein dimers, which are cleaved by proprotein convertases to release the active C-terminal ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct forms of active ligand. Cleavage at the S1 or atypical S0 site produces Gbb15, the conventional small BMP ligand, while cleavage at the NS site produces the larger Gbb38 ligand (1, 2). Here, we found that blocking NS cleavage increased association of the full length prodomain with Gbb15 resulting in a concomitant decrease in signaling activity. NS cleavage is required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not in cell culture to enable Gbb15-Dpp het-erodimer activity. Gbb NS cleavage is also required in vivo for the regulation of pupal ecdysis and viability that is dependent on the type II receptor Wishful thinking (Wit). We found that the ability of Gbb38 to signal requires the expression of either Wit or the type I receptor, Saxophone (Sax). Finally, we discovered that the production of Gbb38 in 3rd instar larvae results when processing at the S1/S0 site is blocked by O-linked glycosylation. Our findings demonstrate that BMP prodomain cleavage can ensure that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligand types that signal preferentially through different receptors and exhibit specific developmental functions.

biochemistry↗

bioRxiv2017

Characterization Of Amylase And Protease Activities In The Digestive System Of Concholepas concholepas (Gastropoda, muricidae)

Concholepas concholepas (loco) is a carnivorous gastropod that inhabits the coast of Chile and Peru. Its fisheries showed a great importance in the past decades, however, now mainly relies on artisanal management of wild stocks. Feeding is one of the important factors that have restrained the establishment of large scale field rearing of loco. C. concholepas food preferences consist of mytilids and cirripeds, however its digestive physiology has not been studied and its digestive enzymes have not been yet characterized. The purification of amylase and protease from the digestive gland and the gland of Leiblein of C. concholepas were performed by ionic exchange chromatography (DEAE-cellulose), and substrate-PAGE indicated the presence of the amylase and protease in the fractions collected from the column. Amylase enzymatic assays showed its maximum activity at pH 7.0 and 50{degrees}C in the digestive gland. Protease on the other hand showed a great acidic activity, specifically at pH 3.0 in both organs, also at 50{degrees}C. Inhibition of the amylolytic activity was observed in the presence of EDTA, and metal ions like MnCl2, MgCl2, ZnSO4, while it was enhanced in the presence of CaCl2, NaCl, KCl. Protease inhibition assays were also performed evidencing mainly the presence of aspartic proteases and a low but not inexistent presence of serine proteases. Our results provide evidence of important proteolytic but also amylolytic activities present in the digestive system of loco, providing evidence that this mollusc has wider digestive capabilities than initially though, which could potentially lead to the development of alternative food diets.

biochemistry↗

bioRxiv2017

Global analysis of plasma lipids identifies liver-derived acyl-carnitines as a fuel source for brown fat thermogenesis

Cold induced thermogenesis is an energy demanding process that protects endotherms against a reduction in ambient temperature. Using non-targeted LC-MS based lipidomics, we identified plasma acylcarnitines as the most significantly changed lipid class in response to the cold. Here we show that acylcarnitines provide fuel for brown fat thermogenesis. In response to the cold, FFAs released from adipocytes activate the nuclear receptor HNF4 to stimulate the expression of genes involved in acylcarnitine metabolism in the liver. Conditional deletion of HNF4 in hepatocytes blocks the cold-induced changes in hepatic gene expression, lowering circulating long chain acylcarnitine (LCAC) levels, and impairing their ability to adapt to the cold. Finally, a bolus of L-carnitine or palmitoylcarnitine rescues the cold sensitivity seen with aging. Our data highlights an elegant mechanism whereby white adipose tissue provides FFAs for hepatic carnitilation to generate plasma LCAC as a fuel source for BAT thermogenesis.\n\nHighlightsO_LIBlood acylcarnitine levels increase in response to the cold.\nC_LIO_LIFFA mobilization in response to the cold activates hepatic HNF4 and stimulates genes involved in acylcarnitine metabolism.\nC_LIO_LIBrown adipocytes metabolize palmitoylcarnitine.\nC_LIO_LICarnitine administration improves thermogenic response in aged mice.\nC_LI\n\nETOCSimcox et al identified acylcarnitines as a novel source of energy for thermogenesis. In response to the cold, the liver activates a transcriptional program through the transcription factor HNF4, leading to increased acylcarnitine levels. They also find that aging mice have reduced acylcarnitine levels and an impaired thermogenic response in the cold. Increasing acylcarnitine levels in old mice increases their ability to adapt to the cold. Their studies discover a physiological role for acylcarnitines in thermogenesis.\n\nGraphical AbstractCold exposure stimulates the sympathetic nervous system to release noradrenaline (NA). Activation of {beta}3-adrenergic receptors stimulates FFA release and activation of the transcription factor HNF4 in the liver. This leads to increased gene expression of enzymes involved in acylcarnitine metabolism. The acylcarnitines are released in the blood to provide fuel for brown fat thermogenesis. These studies highlight the role of the liver in the thermogenic response.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC=\"FIGDIR/small/132241_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (80K):\norg.highwire.dtl.DTLVardef@1282891org.highwire.dtl.DTLVardef@17f7c7forg.highwire.dtl.DTLVardef@c6b637org.highwire.dtl.DTLVardef@1e4f40d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗