Biochemistry Biochemistry最新文献

P/Q-typevoltage-dependent calcium channels (VDCCs) are involvedin the regulation of neurotransmitter release and in the integrationof dendritic inputs in the central nervous system (CNS). However,their precise localization and protein networks in the CNS remainelusive due to the lack of molecular tools applicable to the delicateand complex three-dimensional environment of the living brain. Inthis study, we synthesized peptide-based probes using ω-agatoxinIVA (AgTX), a specific channel blocker for P/Q-type VDCCs derivedfrom the funnel-web spider Agelenopsis aperta. Thefar-red fluorophore-conjugated AgTX probe allowed the visualizationof P/Q-type VDCCs in mouse cerebellum at subcellular spatial resolutionby CLSM imaging. Moreover, by using a photosensitizer-conjugated AgTX,we successfully conducted photooxidation-driven proximity labelingof P/Q-type VDCCs in fixed mouse brain tissues.

Misfolding and aggregation of islet amyloid polypeptide(IAPP)are associated with the decline of β-cells in type 2 diabetes(T2D). Design of short peptide-based inhibitors of β-sheet-richamyloid plaque formation is challenging due to the dynamic natureof the amyloid species. The key sequences involved in cross-amyloidinteractions shed light on the design of peptide-based inhibitors.Amyloid-β peptide (Aβ) has nearly 50% similarity and 25%identity with IAPP sequences, and their cross-interactions have beensuggested to link T2D pathogenesis with Alzheimer’s disease(AD). Here, we show that a short anionic amphiphilic hexapeptide (DM-P02)containing the Aβ domain (Leu¹⁷–Glu²²) effectively suppresses aggregation and oligomerization of IAPPby predominantly interacting with its cationic N-terminus. DM-P02also binds strongly to IAPP in pancreatic β-cell lines, as confirmedby confocal imaging, and protects them from IAPP aggregation-associatedcytotoxicity. Our study underlines the therapeutic potential of shortpeptides for developing anti-amyloid agents.

Acetohydroxyacid synthase (AHAS) is the first enzymein the branched-chainamino acid biosynthesis pathway and is the target for more than 50commercially deployed herbicides. This enzyme has also been suggestedas a target for the discovery of novel antimicrobial and antifungaltherapeutics. Dehydrophos, a natural product antibiotic, is a potentialnew lead inhibitor of AHASs for drug development, whereby the biologicallyactive component is proposed to be methyl acetylphosphonate (MAP).MAP has been suggested as a pyruvate analog that inhibits ThDP-dependentenzymes, including AHAS. Here, the crystal structure of Saccharomyces cerevisiae AHAS in complex with MAPhas been determined to 2.22 Å resolution, showing that it bindscovalently with ThDP. We have also shown that MAP alone is effectivein inhibiting the growth of uropathogenic Escherichia coli (UPEC) in cell susceptibility assays and thatthis activity is lost when exogenous branched-chain amino acids (BCAAs)are added. This suggests that AHAS is a target for the antibioticactivity of MAP and dehydrophos. Our crystal structure provides astarting point for the rational design of ThDP covalently bound AHASinhibitors that are also structurally different from those that havebeen developed previously as commercial herbicides that inhibit AHAS.

WalKR is a two-component system that regulates cell wallhomeostasisand other processes in low-GC Gram-positive bacteria. It is essentialfor viability in Staphylococcus aureus and regulates genes that encode the autolysins and other criticalproteins. The sensor kinase WalK phosphorylates WalR at the asparticacid residue 53 (D53) in the receiver domain, stimulating promoterDNA binding. Unlike most response regulators, WalR is reported tohave a second kinase, the serine/threonine kinase PknB, which phosphorylatesthe receiver domain at the threonine residue 101. We previously reportedthat a walR mutation that changed T101 to a methionineconferred low-level resistance to vancomycin and greatly increasedsusceptibility to tunicamycin along with several other phenotypictraits. In this work, we demonstrate similarities between pknB null and the WalR_T101M mutants. A combinationof in vitro and in vivo data supportsthe idea that PknB interacts with WalR in living cells (consistentwith similar experiments in Bacillus subtilis) and that this likely involves phosphorylation of WalR at T101.Using in silico modeling, we identified a possibleintermolecular hydrogen bond between the T101 phosphate group andE108. This would have the effect of stabilizing the WalR dimerizationinterface. Consistent with this, we find that T101 is important forWalR dimer formation in vivo. The primary activationof WalR is clearly phosphorylation at D53 by WalK. However, our datasuggest that PknB serves as a second potentiating input stabilizingthe WalR dimer.

Many antiviral and anticancer drugs are based on thestructureof pyrimidine nucleosides. Once phosphorylated, they function by interferingwith either DNA or RNA replication or transcription. The antiviralenzyme, viperin, catalyzes the dehydration of CTP to produce 3′-deoxy-3′,4′-didehydroCTP(ddhCTP), which functions as a natural antiviral nucleotide. Variousdrugs mimic the structure of CTP, and furthermore, viperin is upregulatedin some cancers and autoimmune diseases. Therefore, we examined whethersuch drugs may be substrates for viperin. A panel of eight commerciallyavailable nucleosides, including several clinically used drugs, wereenzymatically converted to their triphosphate forms and investigatedas substrates for viperin. Five of the nucleotides were not substrates,indicating that viperin has generally high specificity for CTP. Twonucleotides, 3′-deoxy-3′-fluoroCTP and cytarabine triphosphateelicited reductive cleavage of SAM and were converted to their dehydratedproducts by viperin. The last compound, gemcitabine triphosphate,elicited reductive cleavage of SAM, but no dehydrated product wasobserved, implying that it activates SAM cleavage without undergoinghydrogen atom abstraction. The implications of these results are discussed.

Life has adapted to extreme environments, such as elevated pH, extreme temperatures, and environments with a lack of oxygen. The biomolecules made by organisms that live in extreme environments must adapt to remain folded and functional. Recent computational studies have shown that rRNA, tRNA, and certain mRNAs adapt in thermophilic organisms through an increased GC content. However, few previous studies have assessed the efficiency of adapted thermophilic RNAs. The work presented herein uses minimal hammerhead ribozymes as a model system to determine the effects of high-temperature adaptation, through increased GC base pairing in the ribozyme secondary structure. Two hammerhead ribozymes were designed based upon the minimal hammerhead 16 (HH16) ribozyme, a well-characterized model system. The mesophilic ribozyme has a moderate GC content (44%) in stems I, II, and III, while the thermophilic ribozyme has nearly double the GC content (76%) in those same stems. Both ribozymes share a conserved catalytic core and loop. Thermal denaturation experiments show that the model thermophilic ribozyme is more thermostable with a higher single (cooperative) melting temperature than the model mesophilic ribozyme. However, thermostability is accompanied by a functional penalty. While at certain temperatures, the thermophilic ribozyme reaches the lower observed rates of the mesophilic ribozyme, the thermophilic ribozyme never reaches the same maximal rate of catalysis as the mesophilic ribozyme.

Geranylgeranyl diphosphate synthase (GGDPS) catalyzes the condensation reaction between two isoprenoid substrates: farnesyl diphosphate (FDP) and isopentenyl pyrophosphate (IPP), to form geranylgeranyl diphosphate (GGDP). Due to the flexible nature of the GGDPS active site and its capacity to bind multiple ligands during the reaction coordinate, understanding the structural changes that enable it to accommodate different ligands and catalyze the reaction is essential. While structures exist of substrate and product-bound GGDPS, a lack of high-resolution apo or Mg2+-only structures prevents complete understanding of the enzyme active site. Here, we report apo and Mg2+-only structures of human GGDPS, investigating the key differences between them and published GGDPS structures relating to the enzymatic cycle. A crystal structure resolved to 1.82 Å, the highest-resolution structure of human GGDPS to date, contains five product-bound subunits and one Mg2+-only subunit. The first cryo-EM structure of apo GGDPS is also reported, which exhibited an open hexamer conformation. Comparing the Mg2+-only and apo structures shows that the active site is posed and ready to bind Mg2+ without significant conformational changes. Comparing Mg2+-only to structures with either of the substrates or product bound, we observe changes in residues that actively obstruct or encourage the binding of certain ligands. We also propose an enzymatic mechanism for the structural states of GGDPS and highlight the possibility of Asp188 as the general base in the condensation reaction. These findings enhance our understanding of GGDPS ligand binding, which will improve the knowledge base for rational, structure-based design of future GGDPS inhibitors.

AprD5 is a member of the NAD⁺-dependent short-chaindehydrogenase/reductase (SDR) superfamily. It participates in theapramycin biosynthetic pathway by catalyzing 4′-oxidation ofUDP-β-d-glucose (UBG) to initiate the assembly of UDP-4′-amino-4′-deoxy-β-d-glucose in partnership with the aminotransferase AprL. Here,we report a 1.9 Å crystal structure of AprD5 in complex withNAD⁺ and its substrate UBG, which reveals a well-orderedternary complex poised for catalysis. The cofactor and substrate areprecisely aligned for hydride transfer by a conserved Ser–Tyr–Lyscatalytic triad, while the nucleotide-sugar is constrained in a singlereactive conformation through an extensive hydrogen-bonding networkand a tight electrostatic clamp on the diphosphate group. Although4′-oxidation is a common initiating step in SDR-catalyzed 4′-epimerizationand 4′,6′-dehydration, the structural features thatdirect these closely related enzymes toward distinct reaction outcomesremain poorly understood. Comparative structural analysis revealsthat AprD5 lacks the catalytic architecture required for dehydrationwhile a rigid phosphate clamp and extensive substrate-anchoring networkrestrict the conformational rearrangements necessary for epimerization.Together, these features suggest how a conserved SDR scaffold is specializedfor dedicated 4′-dehydrogenation and highlight general structuralprinciples that may govern catalytic divergence among nucleotidyl-sugarSDR enzymes.

Rapid and accessible detection of pathogens through diversedetectionmodalities remains highly desirable. Most current pathogen detectionassays rely on a single detection modality. Here, we have used thesame RNA switch-CRISPR platform for detection of multiple pathogenicgenomes through three different detection modalities. In this study,the RNA switch is repurposed as a reprogrammable platform for genomedetection, enabling three orthogonal signal outputs: glucometer-based,fluorescence, and visual. Target pathogen genomes selectively regulatecell-free in vitro translation of β-galactosidasethrough the RNA switch, producing several hundred-fold increases inglucose following lactose hydrolysis while simultaneously generatingfluorescence and colorimetric signals. The platform is readily reprogrammedto recognize conserved genomic regions from five distinct pathogens,with specificity dictated by CRISPR–Cas12a-mediated processingof a DNA regulator that controls RNA switch activation and initiatesthe downstream cell-free translation cascade. Glucometer-based, fluorescence,and paper-based colorimetric outputs each enabled selective detectionof Salmonella typhi, Salmonella enterica, Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), and Campylobacter jejuni.Fluorescence and colorimetric responses were analyzed both qualitativelyand quantitatively using image processing and showed strong agreementwith glucometer measurements obtained using a handheld glucometer,providing a simple and accessible quantitative readout. The platformwas further validated using the complete Listeria monocytogenes genome, achieving a detection sensitivity approaching a single genomecopy per microliter. Together, this reprogrammable CRISPR-RNA switchplatform enables the same detection setup to be programmed for multipletargets and readouts through three different detection modalities,some of which are well suited for resource-limited environments.