Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration

Macchioni, Lara; Chiasserini, Davide; Mezzasoma, Letizia; Davidescu, Magdalena; Orvietani, Pier Luigi; Fettucciari, Katia; Salviati, Leonardo; Cellini, Barbara; Bellezza, Ilaria

doi:10.3390/antiox10010025

Cited by 9 publications

(7 citation statements)

References 54 publications

(7 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The samples were eluted from the column using SDS buffer (2% SDS, 100 mM Tris–HCl pH 7.2, 10 mM DTT). After immunoprecipitation, the protein eluates were precipitated overnight with ice‐cold acetone (1:4 v/v) and subjected to sample preparation for proteomics according to previously published procedures 74 …”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

et al. 2022

Self Cite

View full text Add to dashboard Buy / Rent full text

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT‐Ma) and a minor polymorphic form (AGT‐Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT‐Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT‐Mi. An in‐depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT‐Ma and AGT‐Mi. Finally, co‐immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein–protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well‐defined fitness window, thus expanding the adaptability potential of the protein.

show abstract

Section: Methodsmentioning

confidence: 99%

“…After immunoprecipitation, the protein eluates were precipitated overnight with ice-cold acetone (1:4 v/v) and subjected to sample preparation for proteomics according to previously published procedures. 74…”

Section: Western Blot and Coimmunoprecipitationmentioning

confidence: 99%

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

et al. 2022

Self Cite

View full text Add to dashboard Buy / Rent full text

show abstract

“…Given that early AMD is hallmarked by persistent inflammation [35,36], we investigated if FH might influence the levels of relevant inflammatory cytokines, including interleukin-6 (IL6), C-C Motif Chemokine Ligand 2 (CCL2), and interleukin-8 (CXCL8). When FH was downregulated in hTERT-RPE1 siCFH cells, we observed an upregulation of IL6 (3-fold at 48 h and 2-fold at 144 h) and CCL2 (2-fold at 48 and 2.5-fold at 144 h) (Figure 1f-g).…”

Section: Cfh Loss Leads To Upregulation Of C3 and Inflammatory Cytokines In Rpe Cellsmentioning

confidence: 99%

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

Armento

Schmidt

Sonntag

et al. 2021

IJMS

View full text Add to dashboard Buy / Rent full text

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

show abstract

Section: Reagentsmentioning

confidence: 99%

Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Dindo

Pascarelli

Chiasserini

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Buy / Rent full text

Genetic variations expand the conformational landscape of proteins and may underlie cryptic functions able to influence protein adaptability under unfavorable conditions. Cryptic functions usually associate with regions of increased frustration or even intrinsically disordered, whose role as drivers of innovation is increasingly being recognized. Hence, the balance between protein stability and controlled disorder translates in the dichotomy between conservation and innovability, and should be regarded as the more comprehensive measure of protein fitness. In this context, understanding how genetic variations affect protein fitness is not trivial, since cryptic functions behind frustrated regions are not easily detectable. Herein, we used as model the pyridoxal 5-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population and considered a lower limit of AGT fitness. By solving the structure of AGT-Mi, we could show that three distinct regions, that are structured in AGT-Ma, are disordered in AGT-Mi. Molecular dynamics shows that AGT-Mi samples more flexible conformations than AGT-Ma, supporting a plasticity effect propagated to all the structure. In-depth biochemical characterisation of mutants from a small library of variants encompassing the three regions reproduces the fitness window between AGT-Ma and AGT-Mi. Cellular studies highlight the consequences of this plasticity at functional level and suggest the existence of cryptic functions likely related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between protein stability and frustration to encode cryptic functions that expand the potential innovability of the protein.

show abstract

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration

Cited by 9 publications

References 54 publications

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Contact Info

Product

Resources

About