A structural decryption of cryptochromes.

Cryptochromes (CRYs), which are signaling proteins related to DNA photolyases, play pivotal roles in sensory responses throughout biology, including growth and development, metabolic regulation, circadian rhythm entrainment and geomagnetic field sensing. This review explores the evolutionary relationships and functional diversity of cryptochromes from the perspective of their molecular structures. In general, CRY biological activities derive from their core structural architecture, which is based on a Photolyase Homology Region (PHR) and a more variable and functionally specific Cryptochrome C-terminal Extension (CCE). The α/ß and α-helical domains within the PHR bind FAD, modulate redox reactive residues, accommodate antenna cofactors, recognize small molecules and provide conformationally responsive interaction surfaces for a range of partners. CCEs add structural complexity and divergence, and in doing so, influence photoreceptor reactivity and tailor function. Primary and secondary pockets within the PHR bind myriad moieties and collaborate with the CCEs to tune recognition properties and propagate chemical changes to downstream partners. For some CRYs, changes in homo and hetero-oligomerization couple to light-induced conformational changes, for others, changes in posttranslational modifications couple to cascades of protein interactions with partners and effectors. The structural exploration of cryptochromes underscores how a broad family of signaling proteins with close relationship to light-dependent enzymes achieves a wide range of activities through conservation of key structural and chemical properties upon which function-specific features are elaborated.

Simulating the anti-aggregative effect of fasudil in early dimerisation process of α-synuclein.

The aggregation of the protein α-synuclein into amyloid deposits is associated with multiple neurological disorders, including Parkinson's disease. Soluble amyloid oligomers are reported to exhibit higher toxicity than insoluble amyloid fibrils, with dimers being the smallest toxic oligomer. Small molecule drugs, such as fasudil, have shown potential in targeting α-synuclein aggregation and reducing its toxicity. In this study, we use atomistic molecular dynamics simulations to demonstrate how fasudil affects the earliest stage of aggregation, namely dimerization. Our results show that the presence of fasudil reduces the propensity for intermolecular contact formation between protein chains. Consistent with previous reports, our analysis confirms that fasudil predominantly interacts with the negatively charged C-terminal region of α-synuclein. However, we also observe transient interactions with residues in the charged N-terminal and hydrophobic NAC regions. Our simulations indicate that while fasudil prominently interacts with the C-terminal region, it is the transient interactions with residues in the N-terminal and NAC regions that effectively block the formation of intermolecular contacts between protein chains and prevent early dimerization of this disordered protein.

Molecular Mechanism of pH-Induced Protrusion Configuration Switching in Piscine Betanodavirus Implies a Novel Antiviral Strategy.

Many viruses contain surface spikes or protrusions that are essential for virus entry. These surface structures can thereby be targeted by antiviral drugs to treat viral infections. Nervous necrosis virus (NNV), a simple nonenveloped virus in the genus of betanodavirus, infects fish and damages aquaculture worldwide. NNV has 60 conspicuous surface protrusions, each comprising three protrusion domains (P-domain) of its capsid protein. NNV uses protrusions to bind to common receptors of sialic acids on the host cell surface to initiate its entry via the endocytic pathway. However, structural alterations of NNV in response to acidic conditions encountered during this pathway remain unknown, while detailed interactions of protrusions with receptors are unclear. Here, we used cryo-EM to discover that Grouper NNV protrusions undergo low-pH-induced compaction and resting. NMR and molecular dynamics (MD) simulations were employed to probe the atomic details. A solution structure of the P-domain at pH 7.0 revealed a long flexible loop (amino acids 311-330) and a pocket outlined by this loop. Molecular docking analysis showed that the N-terminal moiety of sialic acid inserted into this pocket to interact with conserved residues inside. MD simulations demonstrated that part of this loop converted to a ß-strand under acidic conditions, allowing for P-domain trimerization and compaction. Additionally, a low-pH-favored conformation is attained for the linker connecting the P-domain to the NNV shell, conferring resting protrusions. Our findings uncover novel pH-dependent conformational switching mechanisms underlying NNV protrusion dynamics potentially utilized for facilitating NNV entry, providing new structural insights into complex NNV-host interactions with the identification of putative druggable hotspots on the protrusion.

Gasdermin D cysteine residues synergistically control its palmitoylation-mediated membrane targeting and assembly.

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.

Structural and Functional Insights into the Delivery Systems of Bacillus and Clostridial Binary Toxins.

Pathogenic Bacillus and clostridial (i.e., Clostridium and Clostridioides) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-type complex composed of a catalytic subunit that is toxic (A) and an oligomeric cell-binding and delivery subunit (B), where upon delivery of A into the cytoplasm of the host cell it catalytically ADP-ribosylates actin and rapidly induces host cell death. In this review, binary toxins expressed by Bacillus thuringiensis, Clostridioides difficile, and Clostridium perfringens will be discussed, with particular focus placed upon the structural elucidations of their respective B subunits and how these findings help to deconvolute how toxic enzyme delivery into target host cells is achieved by these deadly bacteria.

Multiple sclerosis: the NLRP3 inflammasome, gasdermin D, and therapeutics.

Multiple sclerosis (MS) stands as a chronic inflammatory disease characterized by its neurodegenerative impacts on the central nervous system. The complexity of MS and the significant challenges it poses to patients have made the exploration of effective treatments a crucial area of research. Among the various mechanisms under investigation, the role of inflammation in MS progression is of particular interest. Inflammatory responses within the body are regulated by various cellular mechanisms, one of which involves the nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domains (PYD)-containing protein 3 (NLRP3). NLRP3 acts as a sensor within cells, playing a pivotal role in controlling the inflammatory response. Its activation is a critical step leading to the assembly of the NLRP3 inflammasome complex, a process that has profound implications for inflammatory diseases like MS. The NLRP3 inflammasome's activation is intricately linked to the subsequent activation of caspase 1 and gasdermin D (GsdmD), signaling pathways that are central to the inflammatory process. GsdmD, a prominent member of the Gasdermin protein family, is particularly noteworthy for its role in pyroptotic cell death, a form of programmed cell death that is distinct from apoptosis and is characterized by its inflammatory nature. This pathway's activation contributes significantly to the pathology of MS by exacerbating inflammatory responses within the nervous system. Given the detrimental effects of unregulated inflammation in MS, therapeutics targeting these inflammatory processes offer a promising avenue for alleviating the symptoms experienced by patients. This review delves into the intricacies of the pyroptotic pathways, highlighting how the formation of the NLRP3 inflammasome induces such pathways and the potential intervention points for therapeutic agents. By inhibiting key steps within these pathways, it is possible to mitigate the inflammatory response, thereby offering relief to those suffering from MS. Understanding these mechanisms not only sheds light on the pathophysiology of MS but also paves the way for the development of novel therapeutic strategies aimed at controlling the disease's progression through the modulation of the body's inflammatory response.

Diabetic cardiomyopathy: Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis.

Recently, the roles of pyroptosis, a form of cell death induced by activated NOD-like receptor protein 3 (NLRP3) inflammasome, in the pathogenesis of diabetic cardiomyopathy (DCM) have been extensively investigated. However, most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models, and whether various medications and natural products prevent the development of DCM, associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis. The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies, with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors. We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link, given that several studies have provided both direct and indirect evidence under specific conditions. This editorial emphasizes that the current investigation should be circumspect in its conclusion, i.e., not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models. Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM, targeting these biomarkers.

Exploiting UPO versatility to transform rutin in more soluble and bioactive products.

The discovery of unspecific peroxygenases (UPOs) completely changed the paradigm of enzyme-based oxyfunctionalization reactions, as these enzymes can transform a wide variety of substrates with a relatively simple reaction mechanism. The fact that UPO can exert both peroxygenative and peroxidative activity in either aromatic or aliphatic carbons, represents a great potential in the production of high value-added products from natural antioxidants. In this work, the flavonoid rutin has been considered as possible substrate for UPO from Agrocybe aegerita, and its peroxygenation or its peroxidation and successive oligomerization have been studied. Different experiments were performed in order to reduce the range of process variables involved and gaining insight on the behavior of this enzyme, leading to a multivariable optimization of UPO-based rutin modification. While trying to preserve enzyme activity this optimization aimed for maximizing the production of more soluble antioxidants. Reusability of the enzyme was evaluated recovering UPO using an enzymatic membrane reactor, revealing challenges in enzyme stability due to inactivation during the filtration stages. The influence of the radical scavenger ascorbic acid on product formation was investigated, revealing its role in directing the reaction towards hydroxylated rutin derivatives, hence indicating a shift towards more soluble and bioactive products.

Myocardial ischemia-reperfusion injury: The balance mechanism between mitophagy and NLRP3 inflammasome.

Myocardial ischemia-reperfusion injury (MIRI) is an injury to cardiomyocytes due to restoration of blood flow after myocardial infarction (MI). It has recently gained much attention in clinical research with special emphasis on the roles of mitochondrial autophagy and inflammation. A mild inflammatory response promotes recovery of post-ischemic cardiomyocyte function and vascular regeneration, but a severe inflammatory response can cause irreversible and substantial cellular damage. Similarly, moderate mitochondrial autophagy can help inhibit excessive inflammation and protect cardiomyocytes. However, MIRI is aggravated when mitochondrial function is disrupted, such as inadequate clearance of damaged mitochondria or excessive activation of mitophagy. How to moderately control mitochondrial autophagy while promoting its balance with nucleotide-binding oligomerization structural domain receptor protein 3 (NLRP3) inflammasome activation is critical. In this paper, we reviewed the molecular mechanisms of mitochondrial autophagy and NLRP3 inflammasome, described the interaction between NLRP3 inflammasome and mitochondrial autophagy, and the effects of different signaling pathways and molecular proteins on MIRI, to provide a reference for future research.

Characterization of viroplasm-like structures by co-expression of NSP5 and NSP2 across rotavirus species A to J.

Rotaviruses (RVs) are classified into nine species, A-D and F-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions composed of main building block proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 in uninfected cells leads to the formation of viroplasm-like structures (VLSs). Although morphologically identical to viroplasms, VLSs do not produce viral progeny but serve as excellent tools for studying complex viroplasms. A knowledge gap exists regarding non-RVA viroplasms due to the lack of specific antibodies and suitable cell culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from RVJ, all NSP5 interacted with their cognate NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2. IMPORTANCE: Rotaviruses (RVs) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis.

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

Weiwei Decoction alleviates gastric intestinal metaplasia through the olfactomedin 4/nucleotide-binding oligomerization domain 1/caudal-type homeobox gene 2 signaling pathway.

BACKGROUND: Gastric intestinal metaplasia (IM) is a precancerous lesion that is associated with an elevated risk of gastric carcinogenesis. Weiwei Decoction (WWD) is a promising traditional Chinese herbal formula widely employed in clinical for treating IM. Previous studies suggested the potential involvement of the olfactomedin 4 (OLFM4)/nucleotide-binding oligomerization domain 1 (NOD1)/caudal-type homeobox gene 2 (CDX2) signaling pathway in IM regulation. AIM: To verify the regulation of the OLFM4/NOD1/CDX2 pathway in IM, specifically investigating WWD's effectiveness on IM through this pathway. METHODS: Immunohistochemistry for OLFM4, NOD1, and CDX2 was conducted on tissue microarray. GES-1 cells treated with chenodeoxycholic acid were utilized as IM cell models. OLFM4 short hairpin RNA (shRNA), NOD1 shRNA, and OLFM4 pcDNA were transfected to clarify the pathway regulatory relationships. Protein interactions were validated by co-immunoprecipitation. To explore WWD's pharmacological actions, IM rat models were induced using N-methyl-N'-nitro-N-nitrosoguanidine followed by WWD gavage. Gastric cells were treated with WWD-medicated serum. Cytokines and chemokines content were assessed by enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction. RESULTS: The OLFM4/NOD1/CDX2 axis was a characteristic of IM. OLFM4 exhibited direct binding and subsequent down-regulation of NOD1, thereby sustaining the activation of CDX2 and promoting the progression of IM. WWD improved gastric mucosal histological lesions while suppressing intestinal markers KLF transcription factor 4, villin 1, and MUCIN 2 expression in IM rats. Regarding pharmacological actions, WWD suppressed OLFM4 and restored NOD1 expression, consequently reducing CDX2 at the mRNA and protein levels in IM rats. Parallel regulatory mechanisms were observed at the protein level in IM cells treated with WWD-medicated serum. Furthermore, WWD-medicated serum treatment strengthened OLFM4 and NOD1 interaction. In case of anti-inflammatory, WWD restrained interleukin (IL)-6, interferon-gamma, IL-17, macrophage chemoattractant protein-1, macrophage inflammatory protein 1 alpha content in IM rat serum. WWD-medicated serum inhibited tumor necrosis factor alpha, IL-6, IL-8 transcriptions in IM cells. CONCLUSION: The OLFM4/NOD1/CDX2 pathway is involved in the regulation of IM. WWD exerts its therapeutic efficacy on IM through the pathway, additionally attenuating the inflammatory response.

Lyssavirus matrix protein inhibits NLRP3 inflammasome assembly by binding to NLRP3.

Lyssavirus is a kind of neurotropic pathogen that needs to evade peripheral host immunity to enter the central nervous system to accomplish infection. NLRP3 inflammasome activation is essential for the host to defend against pathogen invasion. This study demonstrates that the matrix protein (M) of lyssavirus can inhibit both the priming step and the activation step of NLRP3 inflammasome activation. Specifically, M of lyssavirus can compete with NEK7 for binding to NLRP3, which restricts downstream apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. The serine amino acid at the 158th site of M among lyssavirus is critical for restricting ASC oligomerization. Moreover, recombinant lab-attenuated lyssavirus rabies (rabies lyssavirus [RABV]) with G158S mutation at M decreases interleukin-1ß (IL-1ß) production in bone-marrow-derived dendritic cells (BMDCs) to facilitate lyssavirus invasion into the brain thereby elevating pathogenicity in mice. Taken together, this study reveals a common mechanism by which lyssavirus inhibits NLRP3 inflammasome activation to evade host defenses.

5,18-Dimesitylorangarin: a Stable Antiaromatic [20]Pentaphyrin(1.0.1.0.0) Displaying Remarkable Oxidative Self-Coupling Reactions.

5,18-Dimesitylorangarin and its BF2 complex were synthesized by double SNAr reaction of 3,5-dibromo-BODIPY with 2-pyrrolydipyrrin as the first examples of meso-aryl-substituted orangarin. These orangarins, delineated as [20]pentaphyrin(1.0.1.0.0), are strongly antiaromatic but rather stable. The free base orangarin was coupled by oxidation with MnO2 to give a 11,11'-linked dimer, a cyclooctatetraene(COT)-centered trimer, and a spiro-trimer. Fused COT-centered 3H-orangarin dimer was oxidized to the corresponding 2H-orangarin dimer, which was further coupled to give a triply COT-centered 2H-orangarin tetramer. 3H-Orangarin oligomers are all antiaromatic as evinced by extremely low-field-shifted 1H NMR signals of the inner NH and ill-defined absorption spectra with broad tails. In contrast, COT-centered 2H-orangarin dimer and tetramer show moderately low-field-shifted NH signals and intense NIR absorbance over 900 nm, suggesting effective π-conjugation through the COT bridge and almost non-antiaromatic character. These orangarin oligomers exhibit many reversible redox potentials owing to the intramolecular electronic interactions. Regardless of the different aromatic characters, all the orangarin monomers and oligomers exhibit very rapid excited-state decays.

A cryptic homotypic interaction motif of insect STING is required for its antiviral signaling.

Stimulator of interferon genes (STING) mediates innate immune response upon binding to cyclic GMP-AMP (cGAMP). It recruits tank-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3) through its C-terminal tail and facilitates TBK1-dependent phosphorylation of IRF3 via forming STING polymers in mammalian cells. However, the mechanism behind STING-mediated activation of NF-κB transcription factor, Relish, in insect cells is unknown. Our study revealed that insect STING formed oligomers and the cryptic RIP homotypic interaction motif (cRHIM) was required for its oligomerization and its anti-viral functions. Cells expressing cRHIM-deficient mutants exhibited lower levels of anti-viral molecules, higher viral load after viral infection and weak activation of Relish. Moreover, we observed that under cGAMP stimulation, insect STING interacted with IMD, and deletion of the cRHIM motif on either protein prevented this interaction. Finally, we demonstrated that cGAMP enhanced the amyloid-like property of insect STING aggregates by ThT staining. In summary, our research showed that insect STING employed a homotypic motif to form intermolecular interactions that are essential for its antiviral signaling.

Valosin-Containing Protein (VCP)/p97 Oligomerization.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.

TDRD1 phase separation drives intermitochondrial cement assembly to promote piRNA biogenesis and fertility.

The intermitochondrial cement (IMC) is a prominent germ granule that locates among clustered mitochondria in mammalian germ cells. Serving as a key platform for Piwi-interacting RNA (piRNA) biogenesis; however, how the IMC assembles among mitochondria remains elusive. Here, we identify that Tudor domain-containing 1 (TDRD1) triggers IMC assembly via phase separation. TDRD1 phase separation is driven by the cooperation of its tetramerized coiled-coil domain and dimethylarginine-binding Tudor domains but is independent of its intrinsically disordered region. TDRD1 is recruited to mitochondria by MILI and sequentially enhances mitochondrial clustering and triggers IMC assembly via phase separation to promote piRNA processing. TDRD1 phase separation deficiency in mice disrupts IMC assembly and piRNA biogenesis, leading to transposon de-repression and spermatogenic arrest. Moreover, TDRD1 phase separation is conserved in vertebrates but not in invertebrates. Collectively, our findings demonstrate a role of phase separation in germ granule formation and establish a link between membrane-bound organelles and membrane-less organelles.

Neuroprotection by Anethum graveolens (Dill) Seeds and Its Phytocompounds in SH-SY5Y Neuroblastoma Cell Lines and Acellular Assays.

Neurodegeneration diseases (NDs) are a group of complex diseases primarily characterized by progressive loss of neurons affecting mental function and movement. Oxidative stress is one of the factors contributing to the pathogenesis of NDs, including Alzheimer's disease (AD). These reactive species disturb mitochondrial function and accelerate other undesirable conditions including tau phosphorylation, inflammation, and cell death. Therefore, preventing oxidative stress is one of the imperative methods in the treatment of NDs. To accomplish this, we prepared hexane and ethyl acetate extracts of Anethum graveolens (dill) and identified the major phyto-components (apiol, carvone, and dihydrocarvone) by GC-MS. The extracts and major bioactives were assessed for neuroprotective potential and mechanism in hydrogen peroxide-induced oxidative stress in the SH-SY5Y neuroblastoma cell model and other biochemical assays. The dill (extracts and bioactives) provided statistically significant neuroprotection from 0.1 to 30 µg/mL by mitigating ROS levels, restoring mitochondrial membrane potential, reducing lipid peroxidation, and reviving the glutathione ratio. They moderately inhibited acetylcholine esterase (IC50 dill extracts 400-500 µg/mL; carvone 275.7 µg/mL; apiole 388.3 µg/mL), displayed mild anti-Aß1-42 fibrilization (DHC 26.6%) and good anti-oligomerization activity (>40% by dill-EA, carvone, and apiole). Such multifactorial neuroprotective displayed by dill and bioactives would help develop a safe, low-cost, and small-molecule drug for NDs.

H-NS involved in positive regulation of glycerol dehydratase gene expression in Klebsiella pneumoniae 2e.

Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.