Redoxoma

CEPID Redoxoma

RIDC Redoxoma


Proteomic analysis reveals the effects of UVA light on skin cells

Even at low doses, UVA radiation can drive skin photoaging
PorBy Maria Celia Wider
• CEPIDRIDC Redoxoma
28/04/2022
São Paulo, Braszil

Exposure to sunlight leads to premature skin aging. The action of ultraviolet radiation (UV), which is also carcinogenic, has been widely studied. However, the role of ultraviolet-A light (UVA), one of the wavelength ranges of UV radiation, is still poorly understood. Now, for the first time, a study by RIDC Redoxoma scientists reveals the molecular basis of the stress generated by UVA light in a type of skin cell.

Using proteomic analysis, the researchers characterized UVA radiation-induced senescence in primary keratinocytes, the most abundant cells in the skin, as dependent on antioxidant and pro-inflammatory responses. Combining the proteomics approach with the use of machine learning, they also mapped the subcellular protein reorganization in response to UVA light in immortalized keratinocytes. The results were published in two articles in Scientific Reports and iScience.

“Basically, in one of the articles we did more classic proteomics, to look at changes in protein abundance, and in the other, we looked at changes in the subcellular distribution of these proteins, that is, changes related to localization. We saw that changes in proteome abundance were linked to keratinocyte senescence, which is a process very related to aging that will lead to a pro-inflammatory phenotype. When we focused on the short term, for the effects right after radiation, we saw mainly mitochondrial damage,” said Hellen Paula Valerio, first author of the articles. The study was carried out during her doctorate, supervised by professors Paolo Di Mascio and Graziella Eliza Ronsein, both from the Instituto de Química at Universidade de São Paulo (USP) and members of the RIDC Redoxoma.

Of all the ultraviolet radiation emitted by the Sun that reaches the Earth, about 95% corresponds to UVA light and only 5% to UVB light. Although more abundant, UVA light is less energetic than UVB and was recognized as a carcinogen by the World Health Organization (WHO) only in 2009. UVB radiation causes direct DNA damage, while UVA depends on intermediate species such as photoexcited or radical species to generate damage in cells, with an oxidative component in its mechanism of action.

Senescence

Most studies involving UVA light focus on the effects of high doses of radiation on the dermis, the deepest layer of the skin. In this work, the researchers used a single, low dose, equivalent to about 20 minutes of sun exposure, on primary keratinocytes, which account for 80% of the cells in the epidermis, the most superficial layer of the skin, and saw that radiation causes senescence signals in these cells.

Senescent cells do not multiply. According to Hellen Valerio, there is evidence that aged tissues have more senescent cells than young tissues. Furthermore, evidence shows that if senescence is reversed, tissue can rejuvenate.

To observe changes in protein levels, the researchers used an approach based on shotgun proteomics, explains Graziella Ronsein. The cells were irradiated and, 24h later, the proteins were extracted and analyzed by mass spectrometry, always compared with non-irradiated control cells.

Thus, they saw that UVA light promoted a major modification in the proteome - the set of proteins - of keratinocytes. Mainly, they found a greater abundance of antioxidant enzymes and inflammatory mediators, as well as an increase in p16 levels. P16 is a protein important for cell cycle control and is involved in senescence. Biochemical assays confirmed the proteomic data. The HaCaT lineage, considered pre-tumorigenic, does not become senescent when irradiated.

“This is a response that happens in our body as well. When cells are exposed to some kind of stress, they can go into senescence and this can work as a defense mechanism in the sense that this cell does not propagate the damage it has suffered. But the immortalized cell that we used loses this mechanism and continues to proliferate anyway, and this is a cell that already has a genetic predisposition to tumorigenesis,” explains Valerio.

Another interesting result was that irradiated primary keratinocytes secreted molecules that produced effects in non-irradiated HaCaT cells. That is, they induced oxidative stress in neighboring cells, in this case, activating the immune system in pre-tumor keratinocytes (HaCaT cells). “This is very complex. We’re seeing signals between neighboring cells. Then comes the question of whether to work with a single cell in culture or start ’making’ tissues, co-culture cells,” said Paolo Di Mascio.

According to the researchers, these observations offer insights into the cellular mechanisms by which UVA light causes skin aging. Furthermore, it is the first time that this process has been associated with anti-oxidant and pro-inflammatory responses.

Proteomic architecture

Another aspect of the study was the mapping of the subcellular reorganization of the keratinocyte proteome in response to UVA light. For this investigation, the researchers used irradiated HaCat cells and a control group of cells kept in the dark. As Valerio explains, the cells were lysed using a method in which only the plasma membrane is broken, preserving organelle structure and their proteins, and subjected to a series of differential centrifugations with increasing forces. In this way, fractions were separated, representing four subcellular environments: nucleus, mitochondria, cytosol, and secretory organelles. Proteins were then quantified in each fraction and analyzed by mass spectrometry.

From there, using bioinformatics tools and machine learning, the researcher compiled data from the literature regarding the location of proteins in several available banks and validated the fractionation data she had obtained. By comparing irradiated and non-irradiated cells, she saw that part of the proteins had translocated between the organelles. To confirm these observations, the researcher used confocal microscopy, a classic biochemical tool. Of 1,600 proteins quantified and with the subcellular location identified, more than 200 were redistributed in the irradiated cells.

The main result of this proteomic profile was to identify mitochondria as one of the main targets of UVA-induced stress. The researchers showed that UVA light induces mitochondrial fragmentation, regulates redox proteins, and reduces respiratory rate, leading to changes in the overall energy state of cells. Another important result was the demonstration that UVA radiation triggers a classic DNA damage signaling pathway, with an increase in the number of enzymes involved in oxidative damage repair. The data were validated with the comet assay, a technique designed to assess the presence of DNA lesions.

For the authors, this work represents a comprehensive investigation of the subcellular architecture of HaCaT, allowing inferences about dynamic events involving proteins. “Spatial dynamics means a dynamics of changes. These proteins will move from one place to another as a function of the total metabolism of the cell. The point is that we want to see a broader picture of these changes,” explains Di Mascio.

Infrastructure

According to the researchers, in addition to the scientific relevance, this work showed the importance of developing advanced research platforms within the scope of the RIDC Redoxoma. Ronsein highlights “the use, standardization, and optimization of advanced methodologies and tools in both proteomics and bioinformatics, and their application to a relevant biological problem.”

They agree, however, that the biggest problem in this kind of survey is the lack of infrastructure in the country, especially electrical infrastructure. “The mass spectrometer can never be turned off, there can never be energy variation, the air conditioning can never be turned off, and the air humidity needs to be controlled. The device is ultrasensitive and every time the power is turned off, it takes about two weeks to be calibrated,” says Ronsein.

The article “Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light”, by Hellen Paula Valerio, Felipe Gustavo Ravagnani, Angela Paola Yaya Candela, Bruna Dias Carvalho da Costa, Graziella Eliza Ronsein, and Paolo Di Mascio, can be accessed at https://www.cell.com/iscience/fulltext/S2589-0042(22)00363-7

The article A single dose of Ultraviolet-A induces proteome remodeling and senescence in primary human keratinocytes, by Hellen Paula Valerio, Felipe Gustavo Ravagnani, Graziella Eliza Ronsein & Paolo Di Mascio, can be accessed at https://www.nature.com/ articles/s41598-021-02658-5

Proteomic analysis reveals the effects of UVA light on skin cells
Figure: Paolo Di Mascio e Hellen Paula Valerio