Typpioksidia (NO) käsittelevää . Tetrahydropiopteriinin osuudesta NOS-entsyymifunktioissa

 Ihmisen keho tarvitsee  rakenteellisen aminotypen ja siihen kuuluvan aineenvaihdunnan  lisäksi  paljon funktionaalisia typpeä siältäviä ja käsitteleviä  proteiineja. Tässä  keskityn  NO- molekyyliin , typpioksiiin, jota keho tarvitsee  myös.  ennenkin olen kirjoittanut tetrahydrobiopteriinista, joten tässä kertaan asiaa.  Tgerahydrobiopteriini on smantapainen  kuin foolihappo, muta eroaa siitä, että  sitä pystyy keho valmistamaan itse, jos aineenvaihdunta on  optimaali. Foolihapon saanti kuitenkin tukee sen  muodostumsita ja funktiota. MAhdollsiesti se kuitenkin voi olla  myös vitamiini joissain tilanteissa. Ainakin pitää tunnistaa, milloin tetrahydrobiopteriinin  muodostuminen kehosa kompromittoituu ja  siitä  tulee funktionaalinen puutos. 

Full text links

. 2013 Apr;65(4):358-65.

doi: 10.1002/iub.1136. Epub 2013 Feb 26.

Tetrahydrobiopterin in nitric oxide synthase

Jesús Tejero  ¹ , Dennis Stuehr

Affiliations

• PMID: 23441062
• DOI: 10.1002/iub.1136

Tiivistelmä:  Abstract

 Typpioksidisyntaasi (NOS) on  kriittinen entsyymi välittäjäainemolekyylin NO muodostumisessa  L-arginiiniaminohaposta.  NOS-entsyymi vaatii kofaktoriksi tetrahydrobiopteriinia typpioksidimolekyylin  muodostamiseksi. NO-syntaasi on  on yksi harvoista entsyymeistä , joka käyttää juuri tätä kofaktoria . Sen lisäksi tetrahydrobiopteriinin merkitys  NOS-entyymin katalyyttisessä mekanismissakin poikkeaa muiden  entsyymien  katalyyttisistä mekanismeista. NOS-entsyymin  katalyyttisen syklin aikana  tetrahydrobiopteriini muodostaa radikaalilajinsa, joka sitten  taas redusoituu  palauttaen sen tehokkaasti  ennalleen jokaisen NO-synteesisyklin jälkeen.

Tässä katsauksessa tehdään yhteenvetoa  siitä tiedosta, mitä meillä nyukyään on tetrahydrobiopteriinin roolista NOS-entsyymien rakenteessa, fuktiossa ja katalyyttisessä mekanismissa.  

Nitric oxide synthase (NOS) is a critical enzyme for the production of the messenger molecule nitric oxide (NO) from L-arginine. NOS enzymes require tetrahydrobiopterin as a cofactor for NO synthesis. Besides being one of the few enzymes to use this cofactor, the role of tetrahydrobiopterin in NOS catalytic mechanism is different from other enzymes: during the catalytic cycle of NOS, tetrahydrobiopterin forms a radical species that is again reduced, thus effectively regenerating after each NO synthesis cycle. 

In this review, we summarize our current knowledge about the role of tetrahydrobiopterin in the structure, function, and catalytic mechanism of NOS enzymes. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Johdanto, Introduction

 NO-syntaasit, typpioksidin syntetisoijat, ovat  entsyymeitä, jotka katalysoivat  typpioksidin NO muodsotusta arginiini (arg, R) -nimisestä aminohaposta . NO on  tärkeä signaloiva molekyyli , joka osallistuu lukuisiin biologisiin prosesseihin kuten  hermoimpulssien välittämiseen, verisuonten laajenemiseen ja immuunivasteeseen. NOS-entsyymiproteiinit ovat  aktiiveja kun ne ovat homodimeerimuodossa. Yksi monomeeri  käsittää kaksi hyvin selvästi  määriteltävää domeenia. Toinen on N-terminaalinen ( aminoterminaali)  alue. Se on  oxygenaasidomeeni, sitoo hemiä ja L-arginiinia sekä tetrahydrobiopteriinia (H4B, toinen merkintä: BH4). Toinen domeeni on  C-terminaalinen (karboksyyliterminaali)  alue.  Se on reduktaasidomeeni ja sitoo  flaviiniadeniinidinukleotidia (FAD) flaviinimononukleotidia (FMN) ja nikotinamidiadeniinidinukleotidia (NADPH) ( jotka ovat B-vitamiineista kehossa  muodostettuja koentsyymeitä),  ja  toimittaa  elektoroneja  NADPH:sta käsin oxygenaasidomeenilleen.  Molemmat  domeenit voivat ilmentyä erikseen ja ne ovat  myös puhdistettavissa  erikseen ja  niiden ligandien sitomistapa ja reaktiivisuus on selvitetty.  Oxygenaasi ja reduktaasidomeenien välissä on lyhyt  sekvenssi (30-40 aminohapon jakso) ja se antaa sitoutumiskohdan kalmoduliiniproteiinille (CaM). KUVA allaolevassa linkissä Fig.1.

Nitric oxide synthases (NOS, EC 1.14.1.39) are enzymes that catalyze the formation of nitric oxide (NO) from L-arginine. NO is an important signaling molecule that participates in a number of biological processes, including neurotransmission, vasodilation, and immune response (1). NOS proteins are active as homodimers, and each monomer consists of two well-defined domains. The N-terminal oxygenase domain binds heme, L-arginine, and (6R-)5,6,7,8-tetrahydrobiopterin (H₄B). The C-terminal reductase domain binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) and provides electrons to the oxygenase domain from NADPH. Both domains can be expressed and purified separately, and conserve their ligand binding and reactivity. Between the oxygenase and reductase domains, a short sequence (30–40 aminoacids) provides a binding site for calmodulin (CaM) (Fig. 1).

Figure 1

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Schematic mechanism of electron transfer in NOS enzymes. In the presence of H₄B, the oxygenase domains (red ovals) of two NOS monomers are usually in a dimeric form stabilized by the oxygenase (heme) domains. Top, in the absence of CaM, there is limited electron transfer through the NOS reductase domain (rectangles); NADPH reduces FAD via hydride transfer and then electrons are transferred from FAD to FMN. The electron transfer from FMN to the heme is impaired. Bottom, when CaM binds to the NOS it helps stabilize a conformation where the electron transfer from FMN to heme is enabled and the oxygenase domain can catalyze NO synthesis from L-Arginine. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The oxygenase domain of NOS catalyzes the oxidation of L-arginine to L-citrulline and NO in two sequential oxidation steps (Scheme 1). In the first step, L-arginine is hydroxylated to make N^ω-hydroxy-L-arginine (NOHA) in a process that requires one molecule of NADPH and one molecule of oxygen per mol of L-arginine reacted. In the second step, NOHA is oxidized to L-citrulline and NO and 1 molecule of oxygen and 0.5 molecules of NADPH are required.

 

Scheme 1

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NOS converts L-Arginine to L-Citrulline and NO in a two-step process.

NOS converts L-Arginine to L-Citrulline and NO in a two-step process.

The three mammalian isoforms of NOS show high sequence homology but greatly differ in their localization and regulation. The inducible isoform (iNOS) has a high affinity for CaM and is constitutively active; this isoform is mainly regulated at the transcriptional level. Endothelial (eNOS) and neuronal (nNOS) enzymes are constitutively expressed and are reversibly activated by calcium through the binding of Ca²⁺-containing CaM. Related proteins have been discovered in bacteria but consisting of only the oxygenase domain (2). The bacterial NOS enzymes also contain heme but are less stringent in their pterin requirements and can often bind tetrahydrofolate (H₄F) and H₄B with similar affinity. Due to the absence of a connected reductase domain, they must receive electrons from other electron transfer proteins (2-4). The recently discovered NOS from Sorangium cellulosum is a notable exception to this rule (5).

The general biochemistry of NOS enzymes has been extensively reviewed from the biophysical (6-9) and clinical perspective (10). Reviews on H₄B biochemistry have also treated NOS (11, 12). In this review, we will focus on the function of tetrahydrobiopterin in NOS enzymes.

H₄B Requirement for NOS Catalysis

Early studies of NOS enzymes identified H₄B as a necessary cofactor for NO synthesis (13-15) with a stoichiometry of one molecule of H₄B per subunit (16). Nevertheless, the function of H₄B was controversial, with a variety of suggested roles. A redox cycle involving H₄B and H₂B, as in aromatic amino acid hydroxylases was considered, but this would require one H₄B molecule per cycle, in contradiction with experimental data (15, 17). Strong evidence indicated that H₄B enhanced dimer formation (18, 19). However, the evidences of some redox effect did slowly accumulate. Single turnover experiments indicated that the stability of the heme-oxy complex was dependent on H₄B presence, and H₄B was required for the reaction to advance past the formation of a ferrous dioxygen/ferric superoxide complex (20). Other results indicated that H₄B analogs were able to catalyze L-arginine hydroxylation to a variable extent, but all were reduced forms (21). It was even unclear if H₄B was required for the NOHA oxidation step (22). The detection of a H₄B radical (23-26) and the coupling of this radical formation with product formation (26) finally lead to the currently accepted notion—the decay of the ferrous dioxygen/ferric superoxide complex is linked to the formation of a H₄B radical species (8, 9, 27, 28).

H₄B Binding in NOS

Pterin Binding Affinity of NOS

NOS enzymes bind H₄B with high affinity. The presence of significant amounts of H₄B in the enzyme after purification evinces the tight binding of the cofactor by the native protein (13-15). The binding of L-arginine and H₄B shows a synergistic effect: prior binding of L-arginine increases the binding affinity for H₄B and vice versa (21, 29, 30). Mammalian NOS enzymes bind H₄B with affinities in the nM range. H₄B is usually the preferred substrate over H₂B, except for the eNOS enzyme (31). This fact has important implications for human pathology (10, 12, 31). Bacterial NOS enzymes operate in a wide range of K_D values, and sometimes bind H₄F with higher affinity (2, 32, 33). It should be noted that although many other pterins can bind to NOS (see (11) and references therein), only a few H₄B analogs can support NO synthesis (21). The binding affinities for several pterins are shown in Table 1.

Table 1. Dissociation constants for pterin binding to NOS enzymes

Pterin Binding Pocket in Mammalian and Bacterial NOS

Structures of the oxygenase domains for the three eukaryotic NOS isoforms (34-36) and several bacterial NOS proteins (37-39) have been reported. The alignment of the sequences for these NOS proteins indicates the notable similarity between mammalian and bacterial NOS enzymes (Fig. 2). The presence of additional elements in the N-termini of the mammalian proteins can be also noted; these motifs are involved in dimer formation and pterin binding (Figs. 2 and 3). The available structures clearly show that the absence of this N-termini allows bacterial NOS proteins to bind larger pterins (Fig. 3). The conservation of the H₄B binding environments in eNOS, iNOS, and nNOS is remarkable. Moreover, the bacterial NOS proteins also share many similarities in the pterin binding region. H₄B is bound to NOS by an extensive network of hydrogen bonds, with a highly conserved pattern for the three NOS isoforms (34-36) (Fig. 3). Most hydrogen bonds are contributed by the protein but there is also an interaction with the heme group. Several hydrogen bonds are contributed by the protein backbone and the side chain of a conserved Arg residue (Arg375 in mouse iNOS). A conserved tryptophan residue also provides stabilization through a π-stacking interaction (Trp457 in mouse iNOS). Notably both Arg and Trp residues are conserved in mammalian and bacterial NOS proteins. The pterin binding pocket is placed in the oxygenase dimer interface. This location clearly suggest a relationship with the role of H₄B binding on stabilization of the NOS dimer.

Figure 2

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Sequence alignment of mammalian and bacterial NOS proteins. Regions involved in dimerization are shown in yellow; Zn-binding cysteines are shown in green; residues directly binding H₄B or H₄F are shown in red and blue to indicate that they belong to different monomers. Note the additional N-terminus structure for the mammalian NOS proteins, including the N-terminal hook region and the Zn binding motif. nNOS, rat nNOS; iNOS, mouse iNOS eNOS, bovine eNOS; bsNOS, Bacillus subtilis NOS, saNOS, Staphylococcus aureus NOS; gsNOS, Geobacillus stearothermophilus NOS. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

 

Figure 3

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Protein environment of the H₄B/H₄F cofactor. Panels A and B, comparison of the overall structures of iNOS (panel A) and bsNOS (Panel B). Monomers are shown in blue and yellow. The extra N-terminal portion of iNOS, not present in bacterial NOS, is shown in red. Heme (pink), H₄B (H₄F for bsNOS, yellow) and L-Arg (NOHA for bsNOS, blue) are shown as sticks. Panels C and D, detail of the interactions of the H₄B/H₄F cofactor. Panel C, Several iNOS residues and the heme group form a hydrogen bonding network with the H₄B cofactor. Relevant iNOS residues are shown in green and grey; H₄B (yellow), Heme (pink), and the substrate L-Arginine (blue) are shown as sticks. Trp455 and Phe470 are shown in grey color to indicate that they belong to the other monomer. Two water molecules that form H-bonding interactions with H₄B are shown as red spheres. The hydrogen bonding interactions are shown as yellow dashes. Ser112, Ile456, and Trp457 make H-bonds through the main chain carbonyl groups; Arg375 interacts through its side chain. The side chain of Trp457 forms a π-stacking interaction with H₄B. Panel D, Several bsNOS residues and the heme group form a hydrogen bonding network with the H₄F cofactor. Relevant bsNOS residues are shown in green and grey; H₄F (yellow), Heme (pink), and the substrate NOHA (blue) are shown as sticks. Trp323 and Phe338 are shown in grey color to indicate that they belong to the other monomer. The hydrogen bonding interactions are shown as yellow dashes. Thr324 and Trp325 make H-bonds through the main chain carbonyl groups; Arg243 interacts through its side chain. The side chain of Trp325 forms a π-stacking interaction with H₄F. The figure was made using PyMOL (http://www.pymol.org/) and the crystal structure of the mouse iNOSoxy dimer (PDB entry 1NOD (34)) and the bsNOS dimer (PDB entry 1M7Z (38)). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

H₄B Function in NOS Catalysis

H₄B Role in the L-Arginine Hydroxylation Step

Despite initial difficulties to assess the role of H₄B in NOS catalysis, the study of the formation of NOHA from L-arginine yielded increasing evidence of a role for H₄B beyond the H₄B–H₂B cycle of aromatic hydroxylases. The presence of H₄B increases the rate of the Fe^IIO₂ complex decay, a novel role for H₄B (20). Other studies observed that NOHA formation only occurred in the presence of H₄B (40, 41). As previously pointed out, the observation of a H₄B radical formation during the reaction of NOS with L-arginine clearly indicated the active role of H₄B during the formation of NOHA from L-arginine (23-26). The observation of the build-up of an H₄B radical as the ferrous oxygen complex decays was instrumental in establishing a coherent mechanism (26) (Fig. 4).

Figure 4

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Time course of the formation of the oxygen complex (Fe^IIO₂), oxidized heme (Fe^III), NOHA, and H₄B radical during the single turnover of iNOS. Traces for Fe^IIO₂ and Fe^III are determined by stopped-flow experiments; NOHA is determined from rapid-quench experiments; H₄B radical is determined by Electron Paramagnetic Resonance on rapid freeze experiments. Data from (26). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

First steps of the reaction were clear, with the reductase domain of NOS providing the electrons to reduce the heme, and subsequent oxygen binding to form a ferrous-dioxygen complex (Fe^IIO₂). As this species decays with concomitant formation of an H₄B radical, a one electron transfer from H₄B to the heme-oxy complex with the formation of a ferric peroxo species is expected. From this point, different reactions are possible. By analogy with P450 systems, protonation of this species to form the ferric-hydroperoxo species and eventual formation of the Fe^IVO porphyrin radical (Compound I) species was proposed (Fig. 5). In this process, the H₄B radical is still present after L-arginine hydroxylation, and an additional electron—provided by the flavoprotein domain (42)—is needed to return to the initial resting state.

 Figure 5

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Putative mechanism for the hydroxylation of L-Arginine by NOS. Boxes in continuous lines indicate stable species, boxes in dotted lines indicate transient intermediate species. The oxidation state of H₄B and the substrate bound (L-Arginine/NOHA) are shown in the top left and right, respectively, of each species. The transfer of an electron from H₄B to the ferrous-oxy species triggers the formation of a ferric-peroxy species that will capture two protons and eliminate a water molecule to form a compound I-like species that will carry out the hydroxylation of the substrate L-Arginine. Note that the H₄B radical (red) is not reduced after NOHA formation and has to receive one electron from the flavin domain to continue the catalytic cycle. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.

H₄B Role in the NOHA Oxidation Step

The formation of citrulline and NO from NOHA has many similarities with the process of L-arginine oxidation to NOHA and also substantial differences. We must consider that the oxidation of L-arginine to NOHA requires two electrons and two protons, but the reaction of NOHA to citrulline and NO requires one electron, as a two electron donation would lead to the formation of nitroxyl (NO⁻) species as final product. Therefore, it was considered that the electron transfer from H₄B may not be necessary, and even detrimental, for this step.

Extensive evidence further indicates that H₄B is necessary for NOHA oxidation (43-45). Interestingly, reaction of nNOS with NOHA and hydrogen peroxide in the absence of H₄B leads to the formation of nitroxyl, whereas H₄B directs the reaction toward NO instead (46). Therefore, it was proposed that the H₄B radical formed in the first steps of the reaction could actually be reduced by a Fe^II–NO intermediate to produce the observed Fe^III-NO species (24, 46, 47). Later reports are consistent with such model (44, 48-50).

These observations indicate that the requirement for H₄B in the NOHA oxidation step appears to be two-pronged (Fig. 6). First, H₄B it is needed to transfer an electron to the ferrous-dioxygen complex, as for the L-arginine hydroxylation step. Then, after the reaction of the ferric hydroperoxo species with NOHA, a ferrous nitroxyl complex is formed. The H₄B radical will be reduced by this species giving the product ferric–NO complex and regenerating the H₄B. 

Figure 6

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Putative mechanism for the oxidation of NOHA to NO and L-citrulline by NOS. Boxes in continuous lines indicate stable species, boxes in dotted lines indicate transient intermediate species. The oxidation state of H₄B and the substrate bound (NOHA/L-citrulline) are shown in the top left and right, respectively, of each species. The transfer of an electron from H₄B to the ferrous-oxy species triggers the formation of a ferric peroxy species that will evolve to a hydroperoxo species that will react with NOHA to form citrulline and a ferrous nitroxyl species. Subsequent electron transfer from the ferrous nitroxyl species to the H₄B radical will yield the observed Fe^III–NO species. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Effect of Mutations in the H₄B Binding Pocket

The effect of changes in the H₄B binding pocket illustrates the fine tuning of the H₄B reactivity in NOS. The mutation of iNOS Trp457 by Phe or Ala decreases the rate of H₄B radical formation and accelerates its decay. The ferrous-dioxygen complex also decays at a slower rate similar to that of H₄B radical formation, as expected from the mechanism. The amount of H₄B radical stabilized by Trp457Phe and Trp457Ala is around 75% of wild-type enzyme. These changes cause a decreased NOHA yield (51). Similar effects are observed for the nNOS mutants Trp678Phe and Trp678Ala, indicating a conserved role for this residue in the different isoforms (52). NOHA single turnover experiments have been also reported for the iNOS mutants Trp457Phe and Trp457Ala. The mutants show a decrease in the reaction rate, no detectable formation of H₄B radical or Fe^IIINO species, and decreased citrulline yield (53). The mutants are able to produce NO, to a lesser extent than the wild-type enzyme. These observations suggest that the lack of Fe^III–NO species is due to a rate of formation slower than that of NO dissociation from the Fe^III–NO complex, unlike the wild type enzyme where enough Fe^III–NO accumulates to be detected. The mutations cause the Fe^II–O₂ complex to decay at a slower rate; this decrease correlates with a decreased citrulline yield. These results confirm that modification of the H₄B radical stability impacts the reaction of H₄B with the Fe^II–O₂ complex in both reaction steps.

Replacement of iNOS Arg375 by Lys, Asn, or Asp also causes a decrease in the reaction yield. The underlying causes appear more complex in this case. For the Arg375Asn and Arg375Asp, these effects are partly related to impaired formation of the NOS dimer and diminished ability to stabilize the H₄B radical. Alternatively, the Arg375Lys mutant is able to form the H₄B radical at a faster rate than wild-type iNOS. However, Arg375Lys showed lower NOHA yield, decreased NO synthesis, and increased NADPH oxidation. Thus, fast H₄B reactions can lead to increased uncoupling of NADPH oxidation and NO synthesis. These results stress the need for a timely electron transfer for NO synthesis (54).

Conclusions

Our knowledge about H₄B function in NOS has rapidly evolved. In less than 25 years, H₄B has gone from an exotic cofactor of unknown function to a specialized electron donor with a role never seen before in H₄B dependent enzymes. Some questions about H₄B function still remain, the possible role of H₄B as proton donor during catalysis or the exact protonation role of the H₄B species is still discussed (8, 9, 28). The details of the reaction in bacterial NOS, although largely similar to mammalian NOS, can yield some surprises (2, 45).

 Sitaatti  28.4. 2022

Toivoisin uudelleen arviointia tetrahydrobiopteriinin mahdollisesta  obligatorisesta vitamiinimerkityksestä.  esim kombinoituna  foolihappovalmisteisiin.  Esim.  sars-2 cov virus  kiskoi  profyriinejä ja sinkkejä  ja haittasi  raudan aineenavihduntaa. Saattaa olla että se on tehnyt interaktiota NOS-järjestelmään.

Ubikviliini 2 UBQLN2. Mutaatio tässä geenissä linkkiytyy ALS- tautiin.

https://pubmed.ncbi.nlm.nih.gov/30442662/

  2018 Dec 4;115(49):E11485-E11494.

doi: 10.1073/pnas.1811997115. Epub 2018 Nov 15.

Ubiquilin 2 Modulates ALS/FTD-linked FUS-RNA Complex Dynamics and Stress Granule Formation

Elizabeth J Alexander  ^{1   2} , Amirhossein Ghanbari Niaki 

PMID: 30442662
PMCID: PMC6298105
DOI: 10.1073/pnas.1811997115
Free PMC article

Abstract

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS) (FUS gene 16p11.2) . Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.

Keywords: ALS; FTD; FUS; stress granule; ubiquilin 2.

Conflict of interest statement

The authors declare no conflict of interest.

UBQLN2 geeni, NEDD4-sitova proteiini N4BP4 , ALS15

Aliases for UBQLN2 Gene

• GeneCards Symbol: UBQLN2 ²
• Ubiquilin 2 ^{2 3 5}
• N4BP4 ^{2 3 4 5}
• PLIC2 ^{2 3 4 5}
• PLIC-2 ^{2 4 5}
• Chap1 ^{2 4 5}
• Protein Linking IAP With Cytoskeleton 2 ^{3 4}
• Ubiquitin-Like Product Chap1/Dsk2 ^{3 4}
• Ubiquilin-2 ^{3 4}
• CHAP1/DSK2 ^{2 5}
• RIHFB2157 ^{2 5}

• LIC-2 ^{2 5}
• Dsk2 ^{2 5}
• NEDD4 Binding Protein 4 ²
• Nedd4 Binding Protein 4 ³
• DSK2 Homolog ⁴
• HRIHFB2157 ³
• HPLIC-2 ⁴
• ALS15 ³
• CHAP1 ³
• DSK2 ³

External Ids for UBQLN2 Gene

Substantia nigra kerryttää rautaa ja Se-Ferritiini voi olla matala neuroinflammaatiossa

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668412/

Sci Rep. 2017; 7: 14973.

Published online 2017 Nov 2. doi: 10.1038/s41598-017-14721-1

PMCID: PMC5668412

PMID: 29097764

Iron deposition in substantia nigra: abnormal iron metabolism, neuroinflammatory mechanism and clinical relevance

Zhuo Liu,^#1 Hui-cong Shen et al.

Tähän blogiin liitän geenin ZGRF1 , noin 15- 16 GRF sekvenssimotiivia ainakin

Löysin tämän geenin systemaattisesti käydessäni läpi sinkkisormiproteiinien ryhmiä ja ryhmä GRF- tyyppinen sinkkisormiproteiini ZGRF, jossa on 7 proteiinia jhosta yksikään ei ole tgranskriptiotekijä.  Ensimmäin on  aika kookas eikä sille ole varsinanista omaa funktiota. tiedossa. SAen proteiinirakenteessa löytää grf- motiivia  ainakin  16 sekvenssiå ja erittäin paljon p ja q rakenteita jopa  6  tai 7 p ja q peräkkäin joissain  kohdin, sellaisia pätkiä  jotka muistuttavat  vehnän prolamiinia.  Tuli mieleen että se on  jokin hybridimuotoinen ja sen hajoittamisessa voi olla ongelmaa jos on  esim keliakialle tyypillinen geenistö. Voisiko se olla sellainen geni, joka  meioosissa vain  isonee sukupolvesta toiseen. 

Sen esiintymä  on liitetty  multigeenijoukkoon, josta aiheutuu  lapsuuden puhehäiriöitä ja  apraxiaa.  Sopisi  katsoa tämän liittymä  keliakiageeneihin ja alttiuteen, sillä siinä tapauksessa että on keliakiageenistöä,  pitäisi alkaa gluteeniton ravinto jo  varhain  ettei tämä aiheuta aivoperäistä taantumaa, jotain   sakan kertymää jossain. Tämän  proteiinin normaali degradaatiotiekään  ei kai ole  paremmin  esillä.  Otan protei9inin  vielä tässä esiin, vaikka se on pitkä:

 

1. Genetic Candidate Variants in Two Multigenerational Families with Childhood Apraxia of Speech. Peter B, et al. PLoS One, 2016. PMID 27120335, Free PMC Article
2. A Comprehensive Analysis of Common and Rare Variants to Identify Adiposity Loci in Hispanic Americans: The IRAS Family Study (IRASFS). Gao C, et al. PLoS One, 2015. PMID 26599207, Free PMC Article
3. A genome-wide association study on thyroid function and anti-thyroid peroxidase antibodies in Koreans. Kwak SH, et al. Hum Mol Genet, 2014 Aug 15. PMID 24722205, Free PMC Article
4. The full-ORF clone resource of the German cDNA Consortium. Bechtel S, et al. BMC Genomics, 2007 Oct 31. PMID 17974005, Free PMC Article
5. A systematic gene-based screen of chr4q22-q32 identifies association of a novel susceptibility gene, DKK2, with the quantitative trait of alcohol dependence symptom counts. Kalsi G, et al. Hum Mol Genet, 2010 Jun 15. PMID 20332099, Free PMC Article

See all (17) citations in PubMed

See citations in PubMed for homologs of this gene provided by HomoloGene

GeneRIFs: Gene References Into Functions

What's a GeneRIF?

1. Two novel loci IDH1 and ZGRF1 are associated with adiposity. [meta-analysis]
2. Observational study of gene-disease association. (HuGE Navigator)

sä isoformissa on yksi qpqp jakso ja ppp ja qpq , joitain pp ja qq ja qp.

1. NM_001350397.1 → NP_001337326.1  protein ZGRF1 isoform 2
   
   Status: REVIEWED

   Source sequence(s)

   AC023886, AC106864

   Conserved Domains (4) summary

   pfam06839
   Location:1289 → 1333

   zf-GRF; GRF zinc finger

   pfam10382
   Location:4 → 73

   DUF2439; Protein of unknown function (DUF2439)

   pfam13087
   Location:1797 → 1981

   AAA_12; AAA domain

   cl26261
   Location:1591 → 2003

   AAA_11; AAA domain

ORIGIN      
        1 mesqefivly thqkmkkskv wqdgilkith lgnkailydd kgacleslfl kclevkpgdd
       61 lesdrylitv eevkvagaig ivkqnvnkea pelnsrtfis sgrslgcqps glkrkftgfq
      121 gprqvpkkmv imesgesaas heakktgpti fspfcsmppl fptvgkkdvn niladpeniv
      181 tyknrernam dfssvfspsf qinpevlcee nyfcspvnsg nklsdslltn epvkrdslas
      241 hysgvsqnir skaqilallk sessssceel nsemtehfpq kqpqgslkia tkpkyliqqe
      301 ecaemksten lyyqhqsent mrnksrwamy lssqsspihs stvdgndter kpkaqeddvn
      361 snlkdlslqk iiqfvetyae erkkynvdqs vgnndpswnq evkleipsfn essslqvtcs
      421 saendgilse sdiqednkip fnqndkgcik gsvlikenaq evntcgtlek eyeqsesslp
      481 elkhlqiess nnsrisddit dmiseskmdn eslnsihesl snvtqpflev tfnlnnfets
      541 dteeesqesn kisqdseswv kdilvndgns cfqkrsentn ceeiegehlp fltsvsdkpt
      601 vtfpvketlp sqfcdktyvg fdmgickten tgkeieeysd tlsnfesfkw tdavygdnke
      661 dankpiqevr inydfalppn kskginmnlh iphiqnqiae nsnlfsedaq pqpfilgsdl
      721 dkndehvlps tsssdnsvql lntnqnhyec ialdksnthi snslfyplgk khliskdtea
      781 hisepedlgk irspppdhve vetaregkqy wnprnssels glvntisilk slcehstald
      841 sleilkkknt vfqqgtqqty epdsppevrk pfitvvspks phlhkdsqqi lkedevelse
      901 plqsvqfsss gskeetafqa vipkqierkt cdpkvtspee nistlspvst fslnsrdedf
      961 mvefsetslk artlpddlhf lnlegmkksr slenenlqrl sllsrtqvpl itlprtdgpp
     1021 dldshsymin sntyessgsp mlnlceksav lsfsiepedq netffseesr evnpgdvsln
     1081 nistqskwlk yqntsqcnva tpnrvdkrit dgffaeavsg mhfrdtserq sdavnessld
     1141 svhlqmikgm lyqqrqdfss qdsvsrkkvl slnlkqtskt eeiknvlggs tcynysvkdl
     1201 qeisgselcf psgqkiksay lpqrqihipa vfqspahykq tftscliehl nillfglaqn
     1261 lqkalskvdi sfytslkgek lknaennvps chhsqpaklv mvkkegpnkg rlfytcdgpk
     1321 adrckffkwl edvtpgystq egarpgmvls diksiglylr sqkiplyeec qllvrkgfdf
     1381 qrkqygklkk fttvnpefyn epktklylkl srkerssays kndlwvvskt ldfeldtfia
     1441 csaffgpssi neieilplkg yfpsnwptnm vvhallvcna stelttlkni qdyfnpatlp
     1501 ltqyllttss ptivsnkrvs krkfippaft nvstkfells lgatlklase liqvhklnkd
     1561 qataliqiaq mmashesiee vkelqthtfp itiihgvfga gksyllavvi lffvqlfeks
     1621 eaptignarp wkllissstn vavdrvllgl lslgfenfir vgsvrkiakp ilpyslhags
     1681 eneseqlkel halmkedltp tervyvrksi eqhklgtnrt llkqvrvvgv tcaacpfpcm
     1741 ndlkfpvvvl decsqitepa sllpiarfec eklilvgdpk qlpptiqgsd aahengleqt
     1801 lfdrlclmgh kpillrtqyr chpaisaian dlfykgalmn gvteierspl lewlptlcfy
     1861 nvkgleqier dnsfhnvaea tftlkliqsl iasgiagsmi gvitlyksqm yklchllsav
     1921 dfhhpdiktv qvstvdafqg aekeiiilsc vrtrqvgfid sekrmnvalt rgkrhllivg
     1981 nlaclrknql wgrviqhceg redglqhanq yepqlnhllk dyfekqveek qkkksekeks
     2041 kdkshs
//

 Katson isomeerin 1  q ja p esiintymät. 

Paljon on leusiinia, lysiiniä ja seriiniäkin. On G-R-F sekvenssit 

isolla kirjaimella 

 ORIGIN      
        1 mesqefivly thqkmkkskv wqdGilkith lgnkailydd kgacleslfl kclevkpgdd
       61 lesdRylitv eevkvagaig ivkqnvnkea pelnsrtFis sGRslgcqps glkrkfFtGfq
      121 gpRqvpkkmv imesgesaas heakktgpti Fspfcsmppl fptvGkkdvn niladpeniv
      181 tyknRernam dFssvfspsf qinpevlcee nyfcspvnsG nklsdslltn epvkRdslas
      241 hysgvsqnir skaqilallk sessssceel nsemtehFpq kqpqGslkia tkpkyliqqe
      301 ecaemksten lyyqhqsent mRnksrwamy lssqsspihs stvdgndter kpkaqeddvn
      361 snlkdlslqk iiqFvetyae erkkynvdqs vGnndpswnq evkleipsfn essslqvtcs
      421 saendgilse sdiqednkip fnqndkgcik gsvlikenaq evntcgtlek eyeqsesslp
      481 elkhlqiess nnsRisddit dmiseskmdn eslnsihesl snvtqpFlev tfnlnnfets
      541 dteeesqesn kisqdseswv kdilvndGns cfqkRsentn ceeiegehlp Fltsvsdkpt
      601 vtfpvketlp sqfcdktyvG fdmgickten tgkeieeysd tlsnfesfkw tdavygdnke
      661 dankpiqevR inydFalppn kskGinmnlh iphiqnqiae nsnlfsedaq pqpfilgsdl
      721 dkndehvlps tsssdnsvql lntnqnhyec ialdksnthi snslfyplgk khliskdtea
      781 hisepedlgk irspppdhve vetaRegkqy wnprnssels glvntisilk slcehstald
      841 sleilkkknt vFqqGtqqty epdsppevRk pFitvvspks phlhkdsqqi lkedevelse
      901 plqsvqfsss Gskeetafqa vipkqieRkt cdpkpveFqG hqvkgsatsg vmvRghssql
      961 gcsqFpdste yenfmtetpe lpstcmqidf lqvtspeeni stlspvstfs lnsrdedfmv
     1021 efsetslkar tlpddlhfln leGmkksRsl enenlqrlsl lsrtqvplit lprtdgppdl
     1081 dshsyminsn tyessgspml nlceksavls Fsiepedqne tffseesrev npGdvslnni
     1141 stqskwlkyq ntsqcnvatp nRvdkritdg FfaeavsGmh fRdtserqsd avnessldsv
     1201 hlqmikgmly qqrqdFssqd svsrkkvlsl nlkqtsktee iknvlGgstc ynysvkdlqe
     1261 isgselcfps gqkiksaylp qRqihipavf qspahykqtF tscliehlni llfGlaqnlq
     1321 kalskvdisf ytslkgeklk naennvpsch hsqpaklvmv kkegpnkgRl FytcdGpkad
     1381 RckFfkwled vtpGystqeg aRpgmvlsdi ksiglylrsq kiplyeecql lvrkgFdfqr
     1441 kqyGklkkft tvnpefynep ktklylklsR kerssayskn dlwvvsktld Feldtfiacs
     1501 affGpssine ieilplkgyf psnwptnmvv hallvcnast elttlkniqd yfnpatlplt
     1561 qyllttsspt ivsnkRvskr kFippaftnv stkfellslG atlklaseli qvhklnkdqa
     1621 taliqiaqmm ashesieevk elqthtfpit iihgvfgagk syllavvilf fvqlfeksea
     1681 ptignaRpwk llissstnva vdrvllglls lgFenfirvG svRkiakpil pyslhagsen
     1741 eseqlkelha lmkedltpte rvyvrksieq hklgtnrtll kqvrvvgvtc aacpFpcmnd
     1801 lkfpvvvlde csqitepasl lpiarfecek lilvGdpkql pptiqgsdaa hengleqtlf
     1861 dRlclmghkp illrtqyrch paisaiandl FykgalmnGv teieRsplle wlptlcFynv
     1921 kGleqieRdn sFhnvaeatf tlkliqslia sGiagsmigv itlyksqmyk lchllsavdf
     1981 hhpdiktvqv stvdafqgae keiiilscvR trqvgFidse krmnvaltrG kRhllivgnl
     2041 aclrknqlwg rviqhcegre dglqhanqye pqlnhllkdy fekqveekqk kksekekskd
     2101 kshs
//

GRASP12q13.13), Tamaliini , GRP1- assosioitunut proteiini tamaliini (12

https://www.ncbi.nlm.nih.gov/pubmed/12586822/. telineproteiineja ()scaffoldprotein)  assosioitunut PI- fosfolipideihin.

J Biol Chem. 2003 Apr 25;278(17):14762-8. Epub 2003 Feb 13.

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association.

Kitano J¹, Yamazaki Y, Kimura K, Masukado T, Nakajima Y, Nakanishi S.

Official Symbol

TAMALIN

Official Full Name

trafficking regulator and scaffold protein tamalin

Also known as

GRASP

Summary

This gene encodes a protein that functions as a molecular scaffold, linking receptors, including group 1 metabotropic glutamate receptors, to neuronal proteins. The encoded protein contains conserved domains, including a leucine zipper sequence, PDZ domain and a C-terminal PDZ-binding motif. Alternately spliced transcript variants have been observed for this gene.[provided by RefSeq, Dec 2012]

Expression

Broad expression in bone marrow (RPKM 8.3), fat (RPKM 7.8) and 23 other tissues See more

Orthologs

mouse all 

Preferred Names

general receptor for phosphoinositides 1-associated scaffold protein

Names

GRP1 (general receptor for phosphoinositides 1)-associated scaffold protein

general receptor for phosphoinositides 1 associated scaffold protein

Related articles in PubMed

1. Myristoylation restricts orientation of the GRASP domain on membranes and promotes membrane tethering. Heinrich F, et al. J Biol Chem, 2014 Apr 4. PMID 24505136, Free PMC Article
2. The scaffolding protein GRASP/Tamalin directly binds to Dock180 as well as to cytohesins facilitating GTPase crosstalk in epithelial cell migration. Attar MA, et al. BMC Cell Biol, 2013 Feb 26. PMID 23441967, Free PMC Article
3. Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway. Venkataraman A, et al. Cell Biol Int, 2012. PMID 22931251, Free PMC Article
4. Metabotropic glutamate receptor 5, and its trafficking molecules Norbin and Tamalin, are increased in the CA1 hippocampal region of subjects with schizophrenia. Matosin N, et al. Schizophr Res, 2015 Aug. PMID 26048293
5. PDZ-scaffold protein, Tamalin promotes dendritic outgrowth and arborization in rat hippocampal neuron. Mo J, et al. Biochem Biophys Res Commun, 2012 Jun 1. PMID 22569042

See all (19) citations in PubMed

See citations in PubMed for homologs of this gene provided by HomoloGene

GeneRIFs: Gene References Into Functions

What's a GeneRIF?

1. Schizophrenia subjects compared to controls showed a marked increase in CA1 hippocampal Norbin, and Tamalin proteins (47% and 34% respectively), which are endogenous regulators of mGluR5 signalling and trafficking
2. myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.
3. GRASP bridges the guanine nucleotide exchange factors (GEFs) that activate Arf and Rac, thereby promoting Arf-to-Rac signaling By binding to both cytohesin 2/ARNO and Dock180.
4. GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.
5. the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.
6. protein-protein interaction mediated by ARNO coiled-coil domain required for ARNO induced motility; coiled-coil domain promotes assembly of multiprotein complex containing ARNO and Dock180; assembly of complex requires coiled-coil domain, GRASP and IPCEF
7. The crystal structures of the autoinhibitory PDZ domain of Tamalin has implications for the regulation of metabotropic glutamate receptor trafficking.

AIM2 inflammasomi ja PSCI, Gasdermiini D aukot

https://www.sciencedirect.com/science/article/pii/S0889159119315156?via%3Dihub

AIM2 inflammasome contributes to brain injury and chronic post-stroke cognitive impairment in mice

Author links open overlay panelHyunhaKim^abcJi SeonSeo^abcSeo-YeonLee^dKi-TaeHa^abceByung TaeChoi^abceYong-IlShin^fYoungJu Yun^eHwa KyoungShin^abce

https://doi.org/10.1016/j.bbi.2020.03.011

https://www.nature.com/articles/s41419-020-2248-z 

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• Article
• Open Access
• Published: 30 January 2020

Inhibition of AIM2 inflammasome activation alleviates GSDMD-induced pyroptosis in early brain injury after subarachnoid haemorrhage

• Bin Yuan,
• Xiao-ming Zhou,
•  

Cell Death & Disease volume 11, Article number: 76 (2020) Cite this article

Abstract

Only a few types of inflammasomes have been described in central nervous system cells. Among these, the absent in melanoma 2 (AIM2) inflammasome is primarily found in neurons, is highly specific and can be activated only by double-stranded DNA. Although it has been demonstrated that the AIM2 inflammasome is activated by poly(deoxyadenylic-deoxythymidylic) acid sodium salt and leads to pyroptotic neuronal cell death, the role of AIM2 inflammasome-mediated pyroptosis in early brain injury (EBI) after subarachnoid haemorrhage (SAH) has rarely been studied. Thus, we designed this study to explore the mechanism of gasdermin D(GSDMD)-induced pyroptosis mediated by the AIM2 inflammasome in EBI after SAH. The level of AIM2 from the cerebrospinal fluid (CSF) of patients with SAH was detected. The pathway of AIM2 inflammasome-mediated pyroptosis, the AIM2/Caspase-1/GSDMD pathway, was explored after experimental SAH in vivo and in primary cortical neurons stimulated by oxyhaemoglobin (oxyHb) in vitro. Then, we evaluated GSDMD-induced pyroptosis mediated by the AIM2 inflammasome in AIM2 and caspase-1- deficient mice and primary cortical neurons generated through lentivirus (LV) knockdown. Compared with that of the control samples, the AIM2 level in the CSF of the patients with SAH was significantly increased. Pyroptosis-associated proteins mediated by the AIM2 inflammasome were significantly increased in vivo and in vitro following experimentally induced SAH. After AIM2 and caspase-1 were knocked down by an LV, GSDMD-induced pyroptosis mediated by the AIM2 inflammasome was alleviated in EBI after SAH. Intriguingly, when caspase-1 was knocked down, apoptosis was significantly suppressed via impeding the activation of caspase-3. GSDMD-induced pyroptosis mediated by the AIM2 inflammasome may be involved in EBI following SAH. The inhibition of AIM2 inflammasome activation caused by knocking down AIM2 and caspase-1 alleviates GSDMD-induced pyroptosis in EBI after SAH...

In recent years, an increased number of studies have indicated that inflammasomes are involved in EBI following SAH. Although many inflammasomes have been identified, only a few have been described and characterised in the central nervous system (CNS)⁴. Absent in melanoma 2 (AIM2), a member of the haemopoietic interferon-inducible nuclear 200 family of proteins, induces the formation of a highly specific type of inflammasome in the neurons that can recognise aberrant double-stranded DNA (dsDNA). AIM2 triggers the formation of inflammasomes that also contain the apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 and that induce the cleavage of caspase-1, the maturation of interleukin-1β (IL-1β) and interleukin-18 (IL-18) and pyroptosis. The AIM2 inflammasome mediates pyroptotic neuronal cell death, as has been shown in vivo by incubating cortical neurons with poly(deoxyadenylic-deoxythymidylic) acid sodium salt, a synthetic dsDNA⁵. However, the role of gasdermin D (GSDMD)-induced pyroptosis mediated by the AIM2 inflammasome in the pathogenesis of EBI after SAH has not been clearly elucidated.

GSDMD, a member of the gasdermin protein family, is highly conserved in mammals, but its function has not yet been clarified. Recently, an increasing body of work has suggested that GSDMD is the inducer of pyroptosis⁶. Activated inflammatory caspases efficiently cleave GSDMD at an aspartate site within the linking loop, which enables the release of the GSDMD N-terminus (GSDMD-N), which suspends its auto-inhibition, triggers pyroptosis and binds to phosphatidylinositol phosphates and phosphatidylserine of the cell membrane inner leaflet to induce membrane pore formation and IL-1β secretion⁷. As demonstrated by the crucial role of pyroptosis in immunity and disease, excessive uncontrolled pyroptosis may be detrimental to the host. However, despite these important functions, the potential effects of GSDMD-induced pyroptosis in EBI are still unknown.
Hence, to better understand the mechanism of GSDMD-induced pyroptosis mediated by the AIM2 inflammasome in EBI following SAH, we used an in vivo mouse model of SAH and in vitro cellular model of SAH with oxyhaemoglobin (oxyHb). We also re-assessed GSDMD-induced pyroptosis in EBI following SAH after respectively interfering with the expression of AIM2 and caspase-1 with lentivirus (LV).

Discussion

In the present study, we studied the possible role of GSDMD-induced pyroptosis mediated by the AIM2 inflammasome in the pathogenesis of EBI after SAH. The main findings can be summarised as follows: (1) Pyroptosis-related proteins mediated by the AIM2 inflammasome were upregulated in the brain temporal cortex after SAH and in primary cortical neurons exposed to oxyHb. The expression levels of AIM2, GSDMD, GSDMD-N, caspase-1, caspase-1 p20 and ASC increased continuously in a time-dependent manner. (2) The results from the immunohistochemistry and immunofluorescence staining showed that AIM2 inflammasome-mediated pyroptosis mainly occurred in brain neurons. (3) The inhibition of AIM2 inflammasome activation by knocking down AIM2 and caspase-1 could suppress GSDMD-induced neuronal pyroptosis by decreasing the expression and activation of the pyroptosis-related proteins that are mediated by the AIM2 inflammasome. (4) Knocking down caspase-1 suppressed not only GSDMD activation-induced neuronal pyroptosis but also caspase-3 activation-induced neuronal apoptosis. These findings suggested, for the first time, that pyroptosis could be involved in EBI after SAH through the AIM2/Caspase-1/GSDMD pathway. Moreover, inhibiting the expression and activation of caspase-1 could alleviate both pyroptosis and apoptosis (Supplementary Figure).
The pathophysiology of SAH is complicated and involves multiple pathogenic mechanisms (e.g., inflammation, oxidative stress, and apoptosis). A growing body of evidence indicates that the inflammatory response plays a vital role in injury expansion and brain damage after SAH, including EBI, vasospasm, and delayed neurological deterioration². In recent years, inflammasomes have been demonstrated to be important participants in EBI after SAH¹⁰. The inflammasome is a cytosolic multimeric signalling complex that responds to invading pathogens and host-derived danger signals such that its activation leads to caspase-1 activation. Inflammasome activation proceeds via the formation of a multimolecular complex containing a receptor, an adaptor such as ASC and the cysteine protease caspase-1¹³. Then, activated caspase-1 triggers the maturation of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptotic cell death. Several nucleotide-binding oligomerisation domain (NOD)-like receptors (NLRs), as well as AIM2-like receptors (ALRs), have been shown to form inflammasomes. Most of the inflammasomes described to date contain an NLR protein, namely, NOD-like receptor containing pyrin domain 1(NLRP1), NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, and NLR- and caspase-activating recruitment domain-containing 4 (NLRC4). However, only a few inflammasomes have been studied in the CNS, namely, NLRP1, NLRP2, NLRP3 and AIM2. Among these inflammasomes, the NLRP3 inflammasome has been the most studied. NLRP3 inflammasome-mediated neuroinflammation is involved in many acute and chronic CNS diseases, including SAH^10,14,15. In SAH, activation of the NLRP3 inflammasome is also essential for the modulation of pro-inflammatory cytokines, and inhibition of the NLRP3 inflammasome by pharmacological treatment can alleviate brain injury after SAH^10,16.
In addition to NLRP3, AIM2 is also an important inflammasome involved in CNS infection and injury¹⁷. In the present study, we collected and analysed CSF samples from patients with or without SAH. The results from the analysis of patient-derived CSF samples revealed that the level of the AIM2 protein in the CSF of the SAH patients was significantly greater than that in the CSF from non-SAH patients. The higher the Hunt-Hess grade was, the higher the level of AIM2 that was found in the CSF. In the experimental SAH model, we also found that the AIM2 inflammasome was activated in vivo at 24 h post-SAH and that in vitro, the AIM2 inflammasome was significantly unregulated in cultured cortical neurons 6 h after incubation with oxyHb and the levels continued to increase for 12 and 72 h. To verify the role of GSDMD-induced pyroptosis mediated by the AIM2 inflammasome in EBI after SAH, LV was used to knock down the expression of AIM2. The results showed that AIM2 inflammasome-mediated pyroptosis was significantly alleviated in EBI following SAH.
As reported, the AIM2 inflammasome could be activated by aberrant dsDNA, including bacterial DNA and viral DNA, from pathogens and hosts and by endogenous self-DNA within the cytosol, damaged DNA within the nucleus, and self-DNA secreted by exosomes¹⁸. Wang et al. detected CSF DNA levels in patients with SAH and found that both nuclear and mitochondrial DNA levels in the CSF were significantly increased in the patients with SAH compared with volunteers, and the CSF nuclear and mitochondrial DNA levels were significantly higher on days 1 and 4¹⁹. Moreover, higher CSF DNA levels were associated with worse outcomes for patients with SAH¹⁹. This result was consistent with the increased AIM2 in the SAH patients in our study. Combined with the results of this study showing that the level of AIM2 in CSF increased significantly within 3 days after SAH, we found that the elevated level of DNA in the CSF after SAH was synchronous and consistent with that of AIM2. Thus, we speculate that pyroptosis mediated by the AIM2 inflammasome is involved in EBI after SAH. This kind of EBI should be considered, to be exact, a secondary brain injury.
Recently, some scholars believe that pyroptosis should be redefined as GSDMD-mediated, rather than caspase-1-mediated, programmed necrosis²⁰. GSDMD was discovered to form a pore and act as an effector for pyroptosis. In GSDMD-deficient cells, pyroptosis cannot be triggered by known canonical inflammasome ligands²¹. GSDMD contains ~480 amino acids in two domains, and the N-terminal gasdermin domain (GSDMD-N) and the C-terminal gasdermin domain (GSDMD-C) are linked by a long loop. Mounting evidence has demonstrated that GSDMD plays a key role in CNS injury^22,23. In our study, we also observed that GSDMD was upregulated after SAH. Although GSDMD is considered the initiator of pyroptosis, GSDMD-N is the direct and sole effector of pyroptosis. Activated caspase-1 or caspase-11 efficiently cleaves GSDMD at a conserved glutamic acid residue (D276 in mouse and D275 in humans)^21,24. The cleaved GSDMD unleashes the pro-pyroptotic N-terminal fragment from the auto-inhibited state maintained by the C-terminus, thus separating the role of GSDMD into that of GSDMD-N and that of GSDMD-C. The released GSDMD-N is oligomerized, and only the oligomerised form of GSDMD-N is able to translocate to the plasma membrane, where it induces cell rupture and the release of inflammatory cytokines, such as IL-1β²⁵. We also observed that the GSDMD-N generated by caspase-1 cleavage forms an oligomer and migrates to the plasma membrane to kill cells.
Furthermore, GSDMD-N alone localises to the plasma membrane, as evidenced by GSDMD-N binding to liposomes in studies in vitro. Binding studies with liposomes and lipid strips revealed that GSDMD-N has a high affinity for liposomes containing lipids such as cardiolipin, phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] compared with other lipid types²⁶. It is through its lipid-binding specificity that GSDMD-N disrupts plasma membranes, which it perpetrates only when exposed to the cytosolic PI-containing inner leaflet but not when exposed to the extracellular outer leaflet, which lacks PI²⁶. While GSDMD-N kills from within the cell, its released form does not harm neighbouring cells. Mounting evidence has shown that the inner ring diameter of the pore formed by GSDMD-N is estimated to be between 10 and 20 nm^6,27,28. Different methods have revealed a pore consisting of 16 or 24 GSDMD-N units^27,28. This finding is consistent with the observation we made in the present study revealing that a large number of pore-like structures appear on the surface of neurons stimulated by oxyHb. The results of the membrane protein analysis suggest that the pore-like structure may be caused by the oligomerization of GSDMD-N at the cell membrane.
In the present study, we also interfered with the expression of caspase-1 by an LV. After caspase-1 was knocked down, GSDMD-induced pyroptosis was markedly reduced in EBI following SAH. Knocking down caspase-1 inhibited not only the expression and activation of GSDMD but also the expression of pyroptosis-related proteins mediated by the AIM2 inflammasome. We propose two explanations for this result: First, caspase-1-mediated inflammatory injury could be greatly alleviated after caspase-1 is knocked down. Caspase-1 is unequivocally required for the proteolytic processing of IL-1β and IL-18; these two cytokines engage their transmembrane receptors IL-1R and IL-18R to promote inflammation via the activation of nuclear factor-kappaB transcriptional programmes that are induced by the activation of myeloid differential protein-88, a key signalling adaptor²⁹. When caspase-1 was knocked down, caspase-1-mediated inflammatory injury may have been alleviated, and the amount of dsDNA released from damaged cells could be reduced. Second, the non-functional AIM2/ASC complex was quickly depleted. Juruj et al. found that a negative feedback loop controlled by ASC/caspase-1 regulates AIM2 complex formation/stability¹³. In the absence of a functional AIM2 inflammasome, the AIM2/ASC complex formed very rapidly. However, this complex was then removed through activated autophagy following AIM2 speck formation¹³. Thus, when caspase-1 levels were deficient, the AIM2 inflammasome was not readily formed and pyroptosis was thus mediated after SAH.
Intriguingly, knocking down caspase-1 alleviated not only GSDMD activation-induced neuronal pyroptosis but also caspase-3 activation-induced neuronal apoptosis. A few other studies have reported that GSDMD-deficient cells may die because of the caspase-1 cleavage of caspase-3/7^21,30. Inhibiting caspase-1 activity may have attenuated caspase-3-dependent apoptosis, but the mechanism remains unclear^31,32,33. We found that caspase-1 was essential for the activation of caspase-3. The cleavage of caspase-3 was significantly hindered when caspase-1 was knocked down in EBI following SAH. However, the expression of caspase-3 was not affected by caspase-1 knockdown. Therefore, we speculated that apoptosis was also alleviated in EBI after SAH, because knocking down caspase-1 hindered the cleavage of caspase-3. It was recently reported that GSDMD was also cleaved during apoptosis. GSDMD was cleaved by caspase-3/7 at D87 during apoptosis, while GSDMD was cleaved by caspase-1 at D275/D276 during pyroptosis³⁴. The p30 N-terminal fragment of GSDMD (GSDMD p30) released by the cleavage at D275/D276 forms pores in the plasma membrane, thereby mediating pyroptotic cell death. Nevertheless, this cleaved GSDMD also generated the p43 fragment of GSDMD (GSDMD p43) at D87 during apoptosis, which inactivated or failed to trigger pyroptosis (Supplementary Figure). We preliminarily found that caspase-1 may be an important protein at the intersection of the pyroptosis and the apoptosis pathways. Inhibition of caspase-1 activity alleviated not only GSDMD activation-induced neuronal pyroptosis but also caspase-3 activation-induced neuronal apoptosis.
In this study, we demonstrated, for the first time, that the GSDMD-induced pyroptosis mediated by the AIM2 inflammasome may be involved in EBI after SAH. When inhibiting the activation of the AIM2 inflammasome by knocking down AIM2 and caspase-1 with LV, the pyroptosis mediated by the AIM2 inflammasome was significantly alleviated. The activation of caspase-3 was also decreased after caspase-1 was knocked down. However, a clearer regulatory mechanism between pyroptosis and apoptosis remains to be further explored.

References