Assessing Cellular Uptake of Exogenous Coenzyme Q10 into Human Skin Cells by X-ray Fluorescence Imaging.

X-ray fluorescence (XRF) imaging is a highly sensitive non-invasive imaging method for detection of small element quantities in objects, from human-sized scales down to single-cell organelles, using various X-ray beam sizes. Our aim was to investigate the cellular uptake and distribution of Q10, a highly conserved coenzyme with antioxidant and bioenergetic properties. Q10 was labeled with iodine (I2-Q10) and individual primary human skin cells were scanned with nano-focused beams. Distribution of I2-Q10 molecules taken up inside the screened individual skin cells was measured, with a clear correlation between individual Q10 uptake and cell size. Experiments revealed that labeling Q10 with iodine causes no artificial side effects as a result of the labeling procedure itself, and thus is a perfect means of investigating bioavailability and distribution of Q10 in cells. In summary, individual cellular Q10 uptake was demonstrated by XRF, opening the path towards Q10 multi-scale tracking for biodistribution studies.

The creatine kinase system in human skin: protective effects of creatine against oxidative and UV damage in vitro and in vivo.

Cutaneous aging is characterized by a decline in cellular energy metabolism, which is mainly caused by detrimental changes in mitochondrial function. The processes involved seem to be predominantly mediated by free radicals known to be generated by exogenous noxes, e.g., solar ultraviolet (UV) radiation. Basically, skin cells try to compensate any loss of mitochondrial energetic capacity by extra-mitochondrial pathways such as glycolysis or the creatine kinase (CK) system. Recent studies reported the presence of cytosolic and mitochondrial isoenzymes of CK, as well as a creatine transporter in human skin. In this study, we analyzed the cutaneous CK system, focusing on those cellular stressors known to play an important role in the process of skin aging. According to our results, a stress-induced decline in mitochondrial energy supply in human epidermal cells correlated with a decrease in mitochondrial CK activity. In addition, we investigated the effects of creatine supplementation on human epidermal cells as a potential mechanism to reinforce the endogenous energy supply in skin. Exogenous creatine was taken up by keratinocytes and increased CK activity, mitochondrial function and protected against free oxygen radical stress. Finally, our new data clearly indicate that human skin cells that are energetically recharged with the naturally occurring energy precursor, creatine, are markedly protected against a variety of cellular stress conditions, like oxidative and UV damage in vitro and in vivo. This may have further implications in modulating processes, which are involved in premature skin aging and skin damage.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology.

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging.

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.

Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor.

The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine.

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.