SWATH-MS Proteomic Analysis of Oxygen-Induced Retinopathy Reveals Novel Potential Therapeutic Targets

Vähätupa, Maria; Nättinen, Janika; Jylhä, Antti; Aapola, Ulla; Kataja, Marko; Kööbi, Peeter; Järvinen, Tero; Uusitalo, Hannu; Uusitalo‐Järvinen, Hannele

doi:10.1167/iovs.18-23831

Cited by 22 publications

(44 citation statements)

References 71 publications

(101 reference statements)

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“…Beyond defense against glaucomatous neurodegeneration by β-cystallin(s), their protective role in type 2 epithelial-to-mesenchymal transition of retinal-pigment epithelial cells occurring in dry age-related macular degeneration has been shown recently [60]. However, all crystallins were found to be upregulated in a neonatal mouse model of oxygen-induced retinopathy [61][62][63][64], which indicated a developmental stage-dependent control of their expression. Nevertheless, our data summarized in Figure 3 support the observation about a highly correlated group of α-, β-, and γ-crystallin genes [65].…”

Section: Discussionmentioning

confidence: 99%

17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics

et al. 2020

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To facilitate the development of broad-spectrum retina neuroprotectants that can be delivered through topical dosage forms, this proteomics study focused on analyzing target engagements through the identification of functional protein networks impacted after delivery of 17β-estradiol in eye drops. Specifically, the retinae of ovariectomized Brown Norway rats treated with daily eye drops of 17β-estradiol for three weeks were compared to those of vehicle-treated ovariectomized control animals. We searched the acquired raw data against a composite protein sequence database by using Mascot, as well as employed label-free quantification to detect changes in protein abundances. Our investigation using rigorous validation criteria revealed 331 estrogen-regulated proteins in the rat retina (158 were up-regulated, while 173 were down-regulated by 17β-estradiol delivered in eye drops). Comprehensive pathway analyses indicate that these proteins are relevant overall to nervous system development and function, tissue development, organ development, as well as visual system development and function. We also present 18 protein networks with associated canonical pathways showing the effects of treatments for the detailed analyses of target engagements regarding potential application of estrogens as topically delivered broad-spectrum retina neuroprotectants. Profound impact on crystallins is discussed as one of the plausible neuroprotective mechanisms.

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Section: Discussionmentioning

confidence: 99%

17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics

et al. 2020

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“…Prior to SWATH analysis method, a relative protein quantification library was built from several data-dependent analysis (DDA) runs with the same LC gradient and instrument settings that were used for SWATH analyses. Before MS runs, samples were subjected to reduction, alkylation, and digestion with calpain protease instead of trypsin as described in detail in Reference [29]. After digestion, peptides were diluted to 14 µl of sample buffer (2% acetonitrile and 0.1% formic acid) and 1 µl of sample was injected to the triple TOF mass spectrometry.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Laajala

Hankaniemi

Määttä

et al. 2019

Viruses

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Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

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“…We tried to quantify by ELISA some of the growth factors implicated in the blood‐derived stimuli of wound closure, but could not detect them from wound lysates, which is in line with the short half‐life of growth factors in the protease‐rich milieu of the wound . Then, we tried a proteomics‐based approach, but no growth factors were among the detected proteins. This finding is in line with the fact that a 30‐kDa dialysis cut‐off value was used in the processing of tissue samples for proteomics .…”

Section: Discussionmentioning

confidence: 99%

“…Then, we tried a proteomics‐based approach, but no growth factors were among the detected proteins. This finding is in line with the fact that a 30‐kDa dialysis cut‐off value was used in the processing of tissue samples for proteomics . Most recent data have shown that R‐Ras is also crucial for lumenization of new capillaries and microvessels in ischaemic conditions .…”

Section: Discussionmentioning

confidence: 99%

R‐Ras regulates vascular permeability, but not overall healing in skin wounds

Ketomäki

Vähätupa

May

et al. 2018

Experimental Dermatology

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Wounds close by keratinocytes migrating from the edge of the wound and re‐epithelializing the epidermis. It has been proposed that the major stimuli for wound closure are blood‐derived growth factors, chemokines and cytokines. The small GTPase R‐Ras, a known integrin activator, also regulates vascular permeability during angiogenesis, and blood vessels lacking R‐Ras leak plasma proteins constantly. We explored whether the access to blood‐derived proteins influences skin wound healing in R‐Ras knockout (KO) mice. In skin wounds, R‐Ras expression was mostly restricted to the vasculature in the granulation tissue. Angiogenic blood vessels in the R‐Ras KO mice were significantly more permeable than in wild‐type (WT) controls. Although the distances between epidermal tongues, and the panniculus carnosus muscles, were significantly longer in R‐Ras KO than WT controls before the granulation tissue formation took place, there were no differences in the wound closure or re‐epithelialization rates or granulation tissue formation. These findings were also corroborated in a special splint excision wound model. Our study shows that although R‐Ras does not influence the skin wound healing itself, the blood vessels lacking R‐Ras are leaky and thus could facilitate the access of blood‐derived proteins to the wound.

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SWATH-MS Proteomic Analysis of Oxygen-Induced Retinopathy Reveals Novel Potential Therapeutic Targets

Cited by 22 publications

References 71 publications

17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics

17β-Estradiol Delivered in Eye Drops: Evidence of Impact on Protein Networks and Associated Biological Processes in the Rat Retina through Quantitative Proteomics

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

R‐Ras regulates vascular permeability, but not overall healing in skin wounds

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