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Molecular & Cellular Proteomics : MCP Dec 2020Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in...
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 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 protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.
Topics: Active Transport, Cell Nucleus; Animals; Autophagy; Cell Nucleus; HEK293 Cells; HeLa Cells; Hep G2 Cells; Hepatocytes; Humans; I-kappa B Proteins; Inflammation; Liver; Male; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Nuclear Pore Complex Proteins; Protein Aggregates; Protein Binding; Protein Multimerization; Protoporphyrins; RNA, Small Interfering; Sequestosome-1 Protein; Solubility
PubMed: 32912968
DOI: 10.1074/mcp.RA120.002316 -
Genetics in Medicine : Official Journal... Jan 2021Erythropoietic protoporphyria (EPP), characterized by painful cutaneous photosensitivity, results from pathogenic variants in ferrochelatase (FECH). For 96% of patients,...
PURPOSE
Erythropoietic protoporphyria (EPP), characterized by painful cutaneous photosensitivity, results from pathogenic variants in ferrochelatase (FECH). For 96% of patients, EPP results from coinheriting a rare pathogenic variant in trans of a common hypomorphic variant c.315-48T>C (minor allele frequency 0.05). The estimated prevalence of EPP derived from the number of diagnosed individuals in Europe is 0.00092%, but this may be conservative due to underdiagnosis. No study has estimated EPP prevalence using large genetic data sets.
METHODS
Disease-associated FECH variants were identified in the UK Biobank, a data set of 500,953 individuals including 49,960 exome sequences. EPP prevalence was then estimated. The association of FECH variants with EPP-related traits was assessed.
RESULTS
Analysis of pathogenic FECH variants in the UK Biobank provides evidence that EPP prevalence is 0.0059% (95% confidence interval [CI]: 0.0042-0.0076%), 1.7-3.0 times more common than previously thought in the UK. In homozygotes for the common c.315-48T>C FECH variant, there was a novel decrement in both erythrocyte mean corpuscular volume (MCV) and hemoglobin.
CONCLUSION
The prevalence of EPP has been underestimated secondary to underdiagnosis. The common c.315-48T>C allele is associated with both MCV and hemoglobin, an association that could be important both for those with and without EPP.
Topics: Biological Specimen Banks; Europe; Ferrochelatase; Humans; Mutation; Protoporphyria, Erythropoietic; United Kingdom
PubMed: 32873934
DOI: 10.1038/s41436-020-00951-8 -
Orphanet Journal of Rare Diseases Aug 2020Erythropoietic protoporphyria (EPP) is an ultra-rare genetic disorder (prevalence 1:150`000) characterized by instant painful phototoxic burn reactions in skin exposed... (Observational Study)
Observational Study
Increased phototoxic burn tolerance time and quality of life in patients with erythropoietic protoporphyria treated with afamelanotide - a three years observational study.
BACKGROUND
Erythropoietic protoporphyria (EPP) is an ultra-rare genetic disorder (prevalence 1:150`000) characterized by instant painful phototoxic burn reactions in skin exposed to visible light. Afamelanotide is the first clinically tested therapy effectively increasing the time EPP patients can spend in direct sunlight without developing symptoms and reducing the number and severity of phototoxic reactions.
OBJECTIVES
We report our data on real-world effectiveness of afamelanotide treatment in EPP and its phototoxic burn protection factor (PBPF).
METHODS
We analysed clinical data collected between 2016 and 2018 in the Swiss EPP cohort (n = 39) on maximum phototoxic burn tolerance time (PBTT), i.e., maximum time spent in sunlight without phototoxic reaction, severity of phototoxic reactions as assessed by an 11-point Likert-type visual analogue scale (VAS), with 0 being no pain and 10 being the worst possible pain, and Quality of Life (QoL), as assessed with an EPP-specific instrument.
RESULTS
Before treatment, the PBTT was median 10 min (IQR 5-20). Under treatment, PBTT increased to median 180 min (IQR 120-240). Individual PBPF increased 1.8- to 180-fold (full range, median 15). The pain severity of the worst phototoxic reaction before treatment was median 10 and under treatment median 6 (IQR 3-7). QoL at the end of the observation period in 2018 (with all the assessed patients under treatment) was 81.4% (IQR 69.4-93.4, n = 34). A 97.4% treatment adherence rate was observed.
CONCLUSION
Treatment of EPP patients with afamelanotide is highly effective under real-world conditions. We suggest PBTT as a clinical meaningful endpoint in further clinical trials.
Topics: Burns; Humans; Protoporphyria, Erythropoietic; Quality of Life; alpha-MSH
PubMed: 32811524
DOI: 10.1186/s13023-020-01505-6 -
The Netherlands Journal of Medicine Jul 2020Porphyrias are rare metabolic disorders. Lack of awareness and knowledge about the clinical features of porphyrias results in diagnostic and therapeutic delays for many... (Review)
Review
Porphyrias are rare metabolic disorders. Lack of awareness and knowledge about the clinical features of porphyrias results in diagnostic and therapeutic delays for many patients. Delays in diagnosing and treating porphyrias can result in severe, progressive morbidity (and mortality) and psychological distress for patients. This review discusses the pathophysiology, diagnosis, treatment, and follow-up of the most prevalent porphyrias: acute intermittent porphyria, porphyria cutanea tarda, and erythropoietic protoporphyria.
Topics: Delayed Diagnosis; Humans; Porphyria Cutanea Tarda; Porphyria, Acute Intermittent; Porphyrias; Practice Guidelines as Topic; Time-to-Treatment
PubMed: 32641543
DOI: No ID Found -
Nature Communications Jun 20205'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel...
5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.
Topics: 5-Aminolevulinate Synthetase; Acyl Coenzyme A; Catalysis; Catalytic Domain; Crystallography, X-Ray; Gene Expression Regulation, Enzymologic; Genetic Diseases, X-Linked; Heme; Humans; Kinetics; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Protein Domains; Protoporphyria, Erythropoietic; Substrate Specificity
PubMed: 32499479
DOI: 10.1038/s41467-020-16586-x -
Annals of Translational Medicine Mar 2020X-linked dominant protoporphyria (XLDPP) is a rare, hereditary disorder that leads to hepatobiliary and hematologic abnormalities including increased erythrocyte...
BACKGROUND
X-linked dominant protoporphyria (XLDPP) is a rare, hereditary disorder that leads to hepatobiliary and hematologic abnormalities including increased erythrocyte protoporphyrin, cutaneous photosensitivity, and decreased iron stores that is caused by a pathogenic mutation of gene.
METHODS
This study aimed to confirm the existence of XLDPP in a Chinese pedigree. We observed and described the dermatoscopic findings of this disorder under dermoscopy, and assessed photo damage in XLDPP patients using the Fotofinder system and very high frequency (VHF) skin ultrasonic system. We performed next generation sequencing and Sanger sequencing to detect and confirm genetic variants in DNA samples from the XLDPP family. Moreover, we monitored the hepatobiliary function as well as hematologic changes in related family members.
RESULTS
As compared to unaffected control subjects, patients exhibited evidence of severe cutaneous photodamage, causing photoaging, an increase in the size of the gallbladder, increased levels of protoporphyrin in red blood cells, an increase in blood levels of uroporphyrin and hematoporphyrin, and iron deficiency.
CONCLUSIONS
XLDPP was validated by the identification of a four-base-pair deletion (c.1706_1709delAGTG, p.E569fs) in (NM_000032.4) in the proband which segregated with the disease in an X-linked dominant pattern, with hemizygous males being more severely affected than heterozygous females. We also found a missense variant in GATA Binding Protein 1 (GATA1).
PubMed: 32355788
DOI: 10.21037/atm.2020.02.80 -
Nucleic Acids Research May 2020Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a...
Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.
Topics: Albumins; Animals; Bone Marrow; COS Cells; Chlorocebus aethiops; Disease Models, Animal; Ferrochelatase; Humans; K562 Cells; Mice; Oligonucleotides; Polymorphism, Single Nucleotide; Protoporphyria, Erythropoietic; RNA Splice Sites; RNA Splicing; Tissue Distribution
PubMed: 32313951
DOI: 10.1093/nar/gkaa229 -
JAMA Dermatology May 2020The effectiveness of afamelanotide treatment in patients with erythropoietic protoporphyria (EPP) in clinical practice who experience pain after light exposure that...
IMPORTANCE
The effectiveness of afamelanotide treatment in patients with erythropoietic protoporphyria (EPP) in clinical practice who experience pain after light exposure that substantially impairs quality of life is unknown.
OBJECTIVE
To evaluate the association of afamelanotide treatment with outcomes in patients with EPP in regular practice during longer-term follow-up.
DESIGN, SETTING, AND PARTICIPANTS
This single-center, prospective postauthorization safety and efficacy cohort study was directed and approved by the European Medicines Agency. Data were collected from patients with EPP treated with afamelanotide at Erasmus MC between June 2016 and September 2018. Analysis began October 2018.
MAIN OUTCOMES AND MEASURES
Time spent outside during treatment, number of phototoxic reactions, disease-specific quality of life, usage of protective clothing, and adverse events.
RESULTS
A total of 117 patients with EPP (59 women [50.4%]; mean [SD] age, 43.0 [15.5] years) were treated with afamelanotide. Nearly all patients continued treatment (115 [98%]) with a median (interquartile range) follow-up of 2.0 (1.3-2.1) years. Compared with baseline, mean time spent outside during treatment increased significantly by an added 6.1 hours per week (95% CI, 3.62-8.67; P < .001). Mean quality of life score improved significantly by 14.01% (95% CI, 4.53%-23.50%; P < .001). Phototoxic reactions were less painful (β, -0.85; 95% CI, -1.43 to -0.26; P < .001), but there was no significant difference in number or duration of reactions. Minor self-limiting adverse events occurred, such as nausea, fatigue, and headache.
CONCLUSIONS AND RELEVANCE
This cohort study found that afamelanotide treatment was associated with improved clinical outcomes and a good safety profile for patients with EPP. The treatment has clinically significant, sustained positive associations with quality of life, is associated with increased duration of sun exposure, and is associated with less severe phototoxic reactions.
Topics: Adolescent; Adult; Aged; Cohort Studies; Dermatologic Agents; Female; Humans; Male; Middle Aged; Prospective Studies; Protoporphyria, Erythropoietic; Quality of Life; Sunlight; Treatment Outcome; Young Adult; alpha-MSH
PubMed: 32186677
DOI: 10.1001/jamadermatol.2020.0352 -
American Journal of Hematology May 2020Reduced ferrochelatase activity in erythropoietic protoporphyria (EPP) causes the accumulation of protoporphyrin IX (PPIX) leading to acute cutaneous photosensitivity...
Reduced ferrochelatase activity in erythropoietic protoporphyria (EPP) causes the accumulation of protoporphyrin IX (PPIX) leading to acute cutaneous photosensitivity and liver injury. Many EPP patients also have a mild hypochromic, microcytic anemia and iron deficiency. Iron deficiency can lead to decreased PPIX accumulation in another erythropoietic porphyria, congenital erythropoietic porphyria (CEP). Expression of the iron regulatory peptide hepcidin is negatively regulated by the serine protease TMPRSS6. Hepcidin induction by siRNA-mediated inhibition of TMPRSS6 expression reduces iron availability and induces iron deficiency. To interrogate the therapeutic potential of iron deficiency to modify EPP, we treated an ethylnitrosourea-induced mouse model of EPP, Fech , with a GalNAc-conjugated Tmprss6 siRNA and PPIX levels, anemia and iron parameters were monitored. The GalNAc-RNAi therapeutic reduces Tmprss6 expression and induces mild iron deficiency in Fech animals. However, decreases in erythrocyte PPIX levels and liver PPIX accumulation were not seen. These results indicate short-term induction of iron deficiency, at least in a murine model of EPP, does not lead to decreased PPIX production.
Topics: Anemia, Iron-Deficiency; Animals; Disease Models, Animal; Female; Humans; Mice; Phenotype; Protoporphyria, Erythropoietic
PubMed: 31990410
DOI: 10.1002/ajh.25743