Metabolic and Regenerative Role of NLRP3 in Response to Thermal Injury

Severe burns are accompanied by hyperinflammation and hypermetabolism, which increase risk of immune compromise, physiologic exhaustion and ultimately, death. However, these post- trauma phenomena are not wholly detrimental and may confer a survival advantage during the acute phase, while chronic alterations contribute to poor outcomes. In particular, chronically enhanced inflammation may interfere with wound healing, increasing risk of infection, fluid loss, impairing thermoregulation and sustaining metabolic dysregulation. Thus, we investigated the beneficial versus injurious role of inflammation after burn focusing on the master regulator of post-trauma inflammation – NLRP3 inflammasome. Although its inflammatory role is established, recent evidence suggests that NLRP3-mediated inflammation has a pivotal effect on metabolism as well. In this study, we investigated NLRP3-mediated inflammation in burn skin and white adipose tissue (WAT). We aimed to determine if 1) local inflammation is beneficial in wound healing, 2) WAT inflammation regulates lipid metabolism (i.e., lipolysis and browning), and whether 3) WAT metabolic alterations in turn impact healing of adjacent skin. In our initial studies, we investigated whether and how NLRP3-mediated inflammation effects post-burn healing. We demonstrated that lack of acute inflammation impairs normal healing, while prolonged inflammation contributes to scarring. Subsequently, we investigated the role of NLRP3-mediated inflammation in WAT metabolism, which can indirectly affect wound healing and substantially impacts post-burn outcomes. Here, we showed that lack of NLRP3-mediated inflammation and altered inflammatory responses enhance lipid turnover acutely and contribute to persistent, augmented browning. Thus, NLRP3 activation has a key upstream role in quelling WAT dysfunction. Finally, we demonstrated that WAT dysfunction and ensuing increases in lipolytic byproducts in turn enhance lipid uptake to injured skin, interfering with normal healing. Taken together, these studies indicated that post-burn inflammation is initially an adaptive response. Acute activation of inflammatory pathways after trauma is not only beneficial in terms of direct inflammatory and immune effects (e.g., cytokine production, macrophage chemotaxis) but also due to regulation of tissue metabolism (e.g., glycolysis, lipolysis, browning). A key mediator of this cross talk between inflammation and metabolism is NLRP3 inflammasome, a critical acute phase mediator that has a more significant role in post-trauma recovery than previously known.Ph.D

Structural studies of P5 and its interactions with BiP and DNAj-containing proteins

Protein disulfide isomerases (PDIs) constitute a family of thiol oxidoreductases that catalyze oxidation, reduction, and isomerization of disulfide bonds in the endoplasmic reticulum (ER). Human P5 (PDIA6) is a PDI family member that affects the prognosis of diseases such as glioblastoma multiforme and hepatocellular carcinoma (HCC). P5 is a protein which contains three domains, catalytically active domains 1 and 2 joined by a flexible linker and a non thiol-reactive domain 3. Here, we determined high-resolution crystal structures of all three P5 domains. The domains all possess a thioredoxin-like structural fold, and the active site of both catalytic domains contains a CGHC motif. The structure of the non-catalytic domain shows a large hydrophobic pocket that could serve as a potential substrate-binding site, similar to PDIA1. Interestingly, this site is blocked by a C-terminal tail that wraps around the non-catalytic domain. This may provide a mechanism of auto-regulation of P5 activity. While its precise function is still unknown, P5 has been shown to interact with BiP, an Hsp70 homolog found in the ER. BiP chaperone prevents protein aggregation and functions in ER quality control. We used pull-down assays, NMR and X-ray crystallography in order to characterize the interaction between P5 and BiP. Pull-downs showed that full-length P5 binds specifically to the nucleotide-binding domain (NBD) of BiP. The affinity is decreased in the presence of ATP, while the binding is not affected by addition of reducing agent. We obtained the crystal structure of the nucleotide-binding domain of BiP in complex with ADP that reveals a conformational switch not observed in previous structures. This switch could mimic structural changes that occur when NBD associates with partner proteins. Interestingly, this region is adjacent to R197, a residue critical for binding to DNAj-type proteins. NMR titrations suggest that P5 and DNAj-type proteins do not compete with each other for BiP binding, but could potentially form a ternary complex. Another less-studied PDI, protein disulfide isomerase-related (PDIR) protein (PDIA5), is a specialized member that participates in the folding of α1-antitrypsin and N-linked glycoproteins. In this thesis, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates. Understanding the molecular mechanisms of folding will facilitate the development of therapeutic strategies because aberrant protein folding has a role in a variety of diseases. Further characterization of P5 and PDIR will provide a greater understanding of the BiP and calnexin/calreticulin folding pathways that are responsible for maintaining ER integrity.Les protéines disulfure isomérases (PDI) constituent une famille de thio-oxydoréductases qui catalysent l'oxydation, la réduction et l'isomérisation des ponts disulfures dans le réticulum endoplasmique (RE). La protéine P5 humaine (PDIA6) est un membre de la famille des PDI qui affecte le pronostic de maladies telles que le glioblastome multiforme et le carcinome hépatocellulaire (CHC). P5 est une protéine qui contient trois domaines fonctionnels. Les domaines catalytiques 1 et 2 qui sont thio-actifs et reliés par une région flexible sont suivis par un troisième domaine non-réactif. Dans ce travail, nous avons déterminé les structures cristallines à haute résolution des trois domaines de la protéine P5. Ils possèdent tous une structure semblable à celle de la thiorédoxine et le site actif de chacun des deux domaines catalytiques contient un motif CGHC. La structure du domaine non catalytique montre une grande poche hydrophobe qui pourrait potentiellement servir de site de liaison pour le substrat, comme dans le cas de PDIA1. Il est intéressant de noter que ce site est bloqué par l'extrémité C-terminale qui s'enroule autour du domaine non-catalytique. Cela pourrait constituer un mécanisme d'auto-régulation de l'activité P5. Alors que sa fonction précise est encore inconnue, il a été montré que P5 pouvait interagir avec BiP, un homologue de la protéine Hsp70 du réticulum endoplasmique (RE). La chaperonne BiP empêche l'agrégation des protéines et a un rôle de contrôle de la qualité dans le RE. Nous avons utilisé des assais d'immunoprécipitation, de resonnance magnétique nucléaire (RMN) et de cristallographie aux rayons X pour caractériser l'interaction entre les protéines P5 et BiP. Les essais d'immunoprécipitation ont montré que la protéine P5 se liait spécifiquement au domaine de de liaison nucléotidique (NBD) de BiP. L'affinité diminue en présence d'ATP alors que la liaison n'est pas affectée par l'addition d'un agent réducteur. Nous avons déterminé la structure cristalline du domaine de liaison nucléotidique de BiP en complexe avec l'ADP qui révèle un changement de conformation qui n'avait pas été observé dans les structures antérieures. Ce changement de conformation pourrait imiter les changements structurels qui se produisent lorsque le NBD s'associe avec d'autres protéines. Il est intéressant de noter que cette région est adjacente au R197, un acide aminé critique pour la liaison aux protéines qui contiennent un domaine DNAj. Les expériences de titrage RMN suggèrent que la protéine P5 et ainsi que celles qui contiennent un domaine DNAj n'entrent pas en concurrence les uns avec les autres pour BiP, mais qu'elles pourraient former un complexe ternaire. Une autre protéine moins étudiée de la famille des PDI, PDIR (PDIA5), est un membre spécialisé qui participe au repliement des protéines α1-antitrypsine et des glycoprotéines N-linked. Durant cette thèse, la structure cristalline du domaine non catalytique de PDIR a été déterminée à une résolution de 1,5 Å. Sa structure adopte un repliement semblable à celui de la thiorédoxine stabilisé par un pont disulfure structurel et une surface de liaison chargée positivement pour les interactions avec les chaperones de l'ER calréticuline et ERp72. Les contacts entre les molécules au sein du cristal imitent potentiellement les interactions de la protéine PDIR avec un substrat mal replié. Les résultats suggèrent que le domaine non catalytique du PDIR joue un rôle clé dans la reconnaissance des protéines partenaires et des substrats.Comme le repliement anormal des protéines est impliqué dans une variété de maladies, la compréhension des mécanismes moléculaires de leur repliement facilitera le développement de stratégies thérapeutiques. Une caractérisation plus poussée de P5 et de PDIR permettra de mieux comprendre les voies de repliement dans lesquelles sont impliquées les protéines BiP et calnexine/calréticuline qui sont responsables du maintien de l'intégrité du réticulum endoplasmique

Structure of the non-catalytic domain of the protein disulfide isomerase-related protein (PDIR) reveals function in protein binding.

Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α1-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates

Crystal contacts identify a putative binding surface for hydrophobic polypeptides.

<p>(A) The Leu143 and Trp144 from the C-terminal tail of the crystallized fragment bind to a pocket in an adjacent PDIR molecule. (B) The base of the pocket is lined with hydrophobic residues, while Glu31 and Arg46 make hydrogen bonds with backbone amide and carbonyl groups.</p

The non-catalytic domain of PDIR contains a conserved positively charged surface.

<p>(A) Mapping of sequence conservation on the surface of the human PDIR domain; invariant residues are colored green. (B) Surface charge; the positively charged (blue) surface coincides with the conserved region. Negative charge is in red. (C) The conserved lysine and arginine residues are located on helices α1 and α3. The domain orientation in panels (B) and (C) is identical to that in the left view of the panel (A).</p

Sequence analysis of the PDIR non-catalytic domain.

<p>(A) Occurrence of the domain in protein disulfide isomerases and other proteins. Human ERp57 is shown for comparison. Catalytic motifs are shown in catalytically-active thioredoxin-like domains. (B) Rooted phylogenetic tree of proteins shown in panel (A). Sequences labeled WUBG_02370 and RNA methyltransferase are proteins from parasitic nematodes <i>Wuchereria bancrofti</i> (EJW86719) and <i>Brugia malayi</i> (XP_001896925); mosquito PDIR is from <i>Aedes aegypti</i> (XP_001659136). The N-terminal catalytic domain of ERp57 was used for the phylogenetic tree. The figure was generated with ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062021#pone.0062021-Thompson1" target="_blank">[29]</a> and TreeViewPPC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062021#pone.0062021-Page1" target="_blank">[30]</a>. (C) Sequence alignment of the non-catalytic domain from PDIR proteins from human (NP_006801), rabbit (XP_002716857), rattlesnake (AFJ50881), chicken (XP_422097), zebrafish (XP_001107048), frog (XP_001086600), fly (XP_609645), and sea urchin (XP_001200801) and the related sequence from <i>Brugia malayi</i> RNA methyltransferase (XP_001896925). The consensus sequence is shown below; the secondary structure elements are above the sequence.</p

The PDIR non-catalytic domain binds to the P-domain of calreticulin.

<p>(A) Downfield region of HSQC spectra of ¹⁵N-labeled P-domain (residues 211–261) titrated with increasing amounts of the non-catalytic domain of PDIR. The spectra show specific chemical shift changes for residues Ile225 and Ile249. (B) Plot of weighted-average ¹H and ¹⁵N chemical shift changes in the ¹⁵N-labeled calreticulin P-domain upon addition of the unlabeled PDIR domain. (C) Mapping of the chemical shifts measured onto the NMR structure of the calreticulin P-domain (PDB code 1k9c). Magenta indicates a large chemical shift change (>0.1 ppm); white indicates no change detected. Residues showing chemical shift changes above 0.07 ppm are labeled. (D) Surface charge representation of the P-domain. Negative charge is shown in red, positive charge is in blue. (E) Titration of the ¹⁵N-labeled P-domain with the PDIR non-catalytic domain in the presence of 0.5 M ammonium sulfate. The overlay corresponds to the P-domain/PDIR molar ratio of 1∶0 (red), 1∶1 (yellow) and 1∶2 (blue). (F) Titration of the ¹⁵N-labeled PDIR non-catalytic domain with increasing amounts of unlabeled P-domain results in shifts and disappearance of a number of peaks. Overlay shows spectra at the PDIR/P-domain molar ratio of 1∶0 (red), 1∶1 (yellow), 1∶2 (cyan), 1∶4 (purple) and 1∶8 (blue).</p

Regulation of glycolysis and the Warburg effect in wound healing

One of the most significant adverse postburn responses is abnormal scar formation, such as keloids. Despite its prolificacy, the underlying pathophysiology of keloid development is unknown. We recently demonstrated that NLRP3 inflammasome, the master regulator of inflammatory and metabolic responses (e.g., aerobic glycolysis), is essential for physiological wound healing. Therefore, burn patients who develop keloids may exhibit altered immunometabolic responses at the site of injury, which interferes with normal healing and portends keloid development. Here, we confirmed keloid NLRP3 activation (cleaved caspase-1 [P &lt; 0.05], IL-1β [P &lt; 0.05], IL-18 [P &lt; 0.01]) and upregulation in Glut1 (P &lt; 0.001) and glycolytic enzymes. Burn skin similarly displayed enhanced glycolysis and Glut1 expression (P &lt; 0.01). However, Glut1 was significantly higher in keloid compared with nonkeloid burn patients (&gt;2 SD above mean). Targeting aberrant glucose metabolism with shikonin, a pyruvate kinase M2 inhibitor, dampened NLRP3-mediated inflammation (cleaved caspase-1 [P &lt; 0.05], IL-1β [P &lt; 0.01]) and improved healing in vivo. In summary, burn skin exhibited evidence of Warburg-like metabolism, similar to keloids. Targeting this altered metabolism could change the trajectory toward normal scarring, indicating the clinical possibility of shikonin for abnormal scar prevention

Data collection and refinement statistics.

1<p>Highest resolution shell is shown in parentheses.</p>2<p>E.S.U.—estimated overall coordinate error based on maximum likelihood.</p>3<p>Stereochemistry was computed using PROCHECK.</p