Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b562scaffold

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

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Faculty Opinions recommendation of Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008090.101756 ◽

2002 ◽

Author(s):

John Shanklin

Keyword(s):

Green Fluorescent Protein ◽

Rational Design ◽

Fluorescent Protein ◽

Bacterial Endotoxin ◽

Protein Mutants ◽

Green Fluorescent

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Influence of meat source, pH and production time on zinc protoporphyrin IX formation as natural colouring agent in nitrite-free dry fermented sausages

Meat Science ◽

10.1016/j.meatsci.2017.08.024 ◽

2018 ◽

Vol 135 ◽

pp. 46-53 ◽

Cited By ~ 9

Author(s):

Hannelore De Maere ◽

Sylvie Chollet ◽

Jos De Brabanter ◽

Chris Michiels ◽

Hubert Paelinck ◽

...

Keyword(s):

Protoporphyrin Ix ◽

Zinc Protoporphyrin ◽

Production Time ◽

Fermented Sausages ◽

Dry Fermented Sausages

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Recognized and Emerging Features of Erythropoietic and X-Linked Protoporphyria

Diagnostics ◽

10.3390/diagnostics12010151 ◽

2022 ◽

Vol 12 (1) ◽

pp. 151

Author(s):

Elena Di Pierro ◽

Francesca Granata ◽

Michele De Canio ◽

Mariateresa Rossi ◽

Andrea Ricci ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Aminolevulinic Acid ◽

Protoporphyrin Ix ◽

Limiting Factor ◽

Zinc Protoporphyrin ◽

Complete Understanding ◽

Inherited Disorders ◽

Rate Limiting ◽

Systemic Accumulation ◽

Made In

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inherited disorders resulting from defects in two different enzymes of the heme biosynthetic pathway, i.e., ferrochelatase (FECH) and delta-aminolevulinic acid synthase-2 (ALAS2), respectively. The ubiquitous FECH catalyzes the insertion of iron into the protoporphyrin ring to generate the final product, heme. After hemoglobinization, FECH can utilize other metals like zinc to bind the remainder of the protoporphyrin molecules, leading to the formation of zinc protoporphyrin. Therefore, FECH deficiency in EPP limits the formation of both heme and zinc protoporphyrin molecules. The erythroid-specific ALAS2 catalyses the synthesis of delta-aminolevulinic acid (ALA), from the union of glycine and succinyl-coenzyme A, in the first step of the pathway in the erythron. In XLP, ALAS2 activity increases, resulting in the amplified formation of ALA, and iron becomes the rate-limiting factor for heme synthesis in the erythroid tissue. Both EPP and XLP lead to the systemic accumulation of protoporphyrin IX (PPIX) in blood, erythrocytes, and tissues causing the major symptom of cutaneous photosensitivity and several other less recognized signs that need to be considered. Although significant advances have been made in our understanding of EPP and XLP in recent years, a complete understanding of the factors governing the variability in clinical expression and the severity (progression) of the disease remains elusive. The present review provides an overview of both well-established facts and the latest findings regarding these rare diseases.

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Inhaled carbon monoxide does not cause pulmonary vasodilation in the late-gestation fetal lamb

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.278.4.l779 ◽

2000 ◽

Vol 278 (4) ◽

pp. L779-L784 ◽

Cited By ~ 15

Author(s):

Theresa R. Grover ◽

Robyn L. Rairigh ◽

Jeanne P. Zenge ◽

Steven H. Abman ◽

John P. Kinsella

Keyword(s):

Carbon Monoxide ◽

Blood Flow ◽

Pulmonary Artery ◽

Vascular Tone ◽

Ductus Arteriosus ◽

Protoporphyrin Ix ◽

Left Lung ◽

Late Gestation ◽

Zinc Protoporphyrin ◽

Pulmonary Vasodilation

As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (INO) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (ICO) may have similar effects on the perinatal lung. To determine whether ICOcan lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of ICOon late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O2fraction < 0.10) to maintain a constant fetal arterial [Formula: see text]. After 60 min (baseline), the lambs were treated with ICO[5–2,500 parts/million (ppm)]. Comparisons were made with INO(5 and 20 ppm) and combined INO(5 ppm) and ICO(100 and 2,500 ppm). We found that ICOdid not alter left lung blood flow or PVR at any of the study doses. In contrast, low-dose INOdecreased PVR by 47% ( P < 0.005). The combination of INOand ICOdid not enhance the vasodilator response to INO. To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that ICOdoes not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus.

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NO, but not CO, attenuates anaphylaxis-induced postsinusoidal contraction and congestion in guinea pig liver

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00648.2002 ◽

2004 ◽

Vol 286 (1) ◽

pp. R94-R100 ◽

Cited By ~ 14

Author(s):

Zonghai Ruan ◽

Toshishige Shibamoto ◽

Tomohiro Shimo ◽

Hideaki Tsuchida ◽

Tomonobu Koizumi ◽

...

Keyword(s):

Guinea Pig ◽

Protoporphyrin Ix ◽

Fold Increase ◽

Liver Weight ◽

Zinc Protoporphyrin ◽

Hepatic Congestion ◽

Occlusion Technique ◽

Venous Resistance ◽

Synthase Inhibitor ◽

Guinea Pig Liver

The pathophysiology of the hepatic vascular response to anaphylaxis in guinea pig is not known. We studied effects of anaphylaxis on hepatic vascular resistances and liver weight in isolated perfused livers derived from guinea pigs sensitized with ovalbumin. We also determined whether nitric oxide (NO) or carbon monoxide (CO) modulates the hepatic anaphylaxis. The livers were perfused portally and recirculatingly at constant flow with diluted blood. With the use of the double-occlusion technique to estimate the hepatic sinusoidal pressure (Pdo), portal venous resistance (Rpv) and hepatic venous resistance (Rhv) were calculated. An antigen injection caused venoconstriction characterized by an increase in Rpv greater than Rhv and was accompanied by a large liver weight gain. Pretreatment with the NO synthase inhibitor NG-nitro-l-arginine methyl ester, but not the heme oxygenase inhibitor zinc protoporphyrin IX, potentiated the antigen-induced venoconstriction by increasing both Rpv and Rhv (2.2- and 1.2-fold increase, respectively). In conclusion, anaphylaxis causes both pre- and postsinusoidal constriction in isolated guinea pig livers. However, the increases in postsinusoidal resistance and Pdo cause hepatic congestion. Endogenously produced NO, but not CO, modulates these responses.

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A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002316 ◽

2020 ◽

Vol 19 (12) ◽

pp. 1968-1985

Author(s):

Yi Liu ◽

Michael J. Trnka ◽

Shenheng Guan ◽

Doyoung Kwon ◽

Do-Hyung Kim ◽

...

Keyword(s):

Protein Aggregation ◽

Nuclear Import ◽

Cell Model ◽

Protoporphyrin Ix ◽

Zinc Protoporphyrin ◽

Primary Hepatocytes ◽

Protein Aggregate ◽

Nuclear Entry ◽

Antibody Recognition ◽

Bona Fide

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

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