Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System.

Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.

LOGGIC/FIREFLY-2: a phase 3, randomized trial of tovorafenib vs. chemotherapy in pediatric and young adult patients with newly diagnosed low-grade glioma harboring an activating RAF alteration.

BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.

A New Method for Albuminuria Measurement Using a Specific Reaction between Albumin and the Luciferin of the Firefly Squid Watasenia scintillans.

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.

A QM/MM Study on the Initiation Reaction of Firefly Bioluminescence-Enzymatic Oxidation of Luciferin.

Among all bioluminescent organisms, the firefly is the most famous, with a high luminescent efficiency of 41%, which is widely used in the fields of biotechnology, biomedicine and so on. The entire bioluminescence (BL) process involves a series of complicated in-vivo chemical reactions. The BL is initiated by the enzymatic oxidation of luciferin (LH2). However, the mechanism of the efficient spin-forbidden oxygenation is far from being totally understood. Via MD simulation and QM/MM calculations, this article describes the complete process of oxygenation in real protein. The oxygenation of luciferin is initiated by a single electron transfer from the trivalent anionic LH2 (L3-) to O2 to form 1[L•2-…O2•-]; the entire reaction is carried out along the ground-state potential energy surface to produce the dioxetanone (FDO-) via three transition states and two intermediates. The low energy barriers of the oxygenation reaction and biradical annihilation involved in the reaction explain this spin-forbidden reaction with high efficiency. This study is helpful for understanding the BL initiation of fireflies and the other oxygen-dependent bioluminescent organisms.

Elucidating the multi-configurational character of the firefly dioxetanone anion and its prototypes in the biradical region using full valence active spaces.

We analyzed the near-degenerate states of the firefly dioxetanone anion (FDO-) and its prototypes, especially in the biradical region, using multi-configurational approaches. The importance of utilizing full valence active spaces by means of density-matrix renormalization group self-consistent field (DMRG-SCF) calculations was described. Our results revealed that the neglect of some valence orbitals can affect the quantitative accuracy in later multi-reference calculations or the qualitative conclusion when optimizing conical intersections. Using all of the relevant valence orbitals of FDO-, we confirmed that there were two conical intersections, as reported in previous work, and that the intersecting states were changed when the active space was enlarged. Beyond these, we found that there were strong interactions between states in the biradical regions, in which the changes in entanglements can be used to visualize the interacting state evolution.

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly, Luciola lateralis.

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Characterization of protein quality control components via dual reporter-containing misfolded cytosolic model substrates.

Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and ß-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast.

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

The spectral-structural relationship of a series of oxyluciferin derivatives.

The absorption and emission spectra of a series of oxyluciferin derivatives with different substituents, as well as 6'-amino oxyluciferins in different enol and keto forms, with or without an active-site model of luciferase, were systematically investigated using density functional theory. The effects of substituents, microenvironment, and the luciferase on the structures, absorption spectra, and fluorescent emission were all taken into account. It was found that a wide range of emission colors can be obtained from various oxyluciferin derivatives with the inclusion of active site residues modeling the luciferase active site. Enol and keto forms are responsible for the emissions observed in experiments. It was suggested that the active site of luciferase must be included in the calculation in order to determine the form of the emitters.

Firefly light flashing: oxygen supply mechanism.

Firefly luminescence is an intriguing phenomenon with potential technological applications, whose biochemistry background was only recently established. The physics side of this phenomenon, however, was still unclear, specifically as far as the oxygen supply mechanism for light flashing is concerned. This uncertainty is due to the complex microscopic structure of the tracheal system: without fully knowing its geometry, one cannot reliably test the proposed mechanisms. We solved this problem using synchrotron phase contrast microtomography and transmission x-ray microscopy, finding that the oxygen consumption corresponding to mitochondria functions exceeds the maximum rate of oxygen diffusion from the tracheal system to the photocytes. Furthermore, the flashing mechanism uses a large portion of this maximum rate. Thus, the flashing control requires passivation of the mitochondria functions, e.g., by nitric oxide, and switching of the oxygen supply from them to photoluminescence.

Vibrational spectra of chemical and isotopic variants of oxyluciferin, the light emitter of firefly bioluminescence.

The chemical complexity of oxyluciferin (OxyLH2), the light-emitting molecule in the bioluminescence of fireflies, originates from the possibility of keto/enol tautomerism and single or double deprotonation. Herein, we present detailed infrared spectroscopic analysis of OxyLH2 and several of its chemical isomers and isotopomers. To facilitate the future characterization of its biogenic forms, we provide accurate assignments of the solid-state and solution FTIR spectra of OxyLH2 based on comparison to six isotopically labeled variants ([2-(13)C]-OxyLH2, [3-(15)N]-OxyLH2, [4-(13)C]-OxyLH2, [5-(13)C]-OxyLH2, [2'-(13)C]-OxyLH2, [3'-(15)N]-OxyLH2), five closely related structural analogues, and theoretically computed spectra. The computed DFT harmonic vibrational force fields (B3LYP and M06 functionals with basis sets of varying flexibility up to 6-311++G**) reproduce well the observed shifts in the IR spectra of both isotopically labeled and structurally related analogues.

Why is firefly oxyluciferin a notoriously labile substance?

The chemistry of firefly bioluminescence is important for numerous applications in biochemistry and analytical chemistry. The emitter of this bioluminescent system, firefly oxyluciferin, is difficult to handle. The cause of its lability was clarified while its synthesis was reinvestigated. A side product was identified and characterized by NMR spectroscopy and X-ray crystallography. The reason for the lability of oxyluciferin is now ascribed to autodimerization of the coexisting enol and keto forms in a Mannich-type reaction.

Firefly Rats: Illuminating the Scientific Community in Transplantation Research.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

On the influence of water on the electronic structure of firefly oxyluciferin anions from absorption spectroscopy of bare and monohydrated ions in vacuo.

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

Theoretical photodynamic study of the photoprotolytic cycle of firefly oxyluciferin.

Firefly oxyluciferin presents a pH-sensitive fluorescence in aqueous solutions. Its fluorescence spectra are composed of two green peaks at different pH values, despite the enolate anion being the only emitter. A computational approach was used to further elucidate the photoprotolytic cycle of oxyluciferin and investigate its pH sensitivity. It was found that oxyluciferin forms π-π stacking complexes both in the ground and excited states, at basic and acidic/neutral pH. However, at different pH values, these complexes adopt a different conformation, which explains the lower energy of the emission at acidic/neutral pH, in comparison with the emission at basic pH.

Oxyluciferin photoacidity: the missing element for solving the keto-enol mystery?

The oxyluciferin family of fluorophores has been receiving much attention from the research community and several systematic studies have been performed in order to gain more insight regarding their photophysical properties and photoprotolytic cycles. In this minireview, we summarize the knowledge obtained so far and define several possible lines for future research. More importantly, we analyze the impact of the discoveries on the firefly bioluminescence phenomenon made so far and explain how they re-open again the discussion regarding the identity (keto or enol species) of the bioluminophore.

Realization of firefly bioluminescence cycle in vitro and in cells.

The quantum yield (Q) of the "cold light" of firefly bioluminescence (BL) is remarkably high due to its nonradiative decay is extremely minimized. Thus, an artificial firefly represents the new generation of biomimetic "cold light" source with highest energy utilization. However, to manufacture a firefly-biomimetic "cold light" in vitro, one has to overcome several challenges including realization of the firefly BL cycle by incorporating the two important enzymes (i.e., firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE)) in one system. Here in this work, using self-prepared Fluc, LRE, and the main substrates, we realized the firefly BL cycle both in vitro and in cells. Moreover, using combinational analyses of HPLC and nESI-CID-MS/MS, we identified the main chemicals in the metabolic pathways underlying the firefly BL cycle. Using theoretical simulations, we revealed an optimum chemical route which balances the reaction cycle to achieve the highest BL intensity with the least chemical supplies. We anticipate that this pioneering study of the firefly cycle would provide industry with the opportunity to design tunable, economical, biomimetic "cold light" device in near future.