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Skp1-cullin-F-box protein (SCF) is a multicomponent E3 ubiquitin (Ub) ligase that ubiquitinates a number of important biologic molecules such as p27, beta-catenin, and IkappaB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG), as well as its family member ROC1/Rbx1, bound to the proinactive form of caspase-3 (pro-caspase-3). Binding was likely mediated through F-box protein, beta-transducin repeat-containing protein (beta-TrCP), which binds to the first 38 amino acids of pro-caspase-3. Importantly, beta-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative beta-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF(beta-TrCP) promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCF(beta-TrCP) E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1), or beta-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCF(beta-TrCP) E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

Tumor cells express lower levels of manganese superoxide dismutase (MnSOD) than their normal counterparts. Enforced expression of MnSOD reverses the malignant phenotype of many transformed cells, suggesting that SOD2 is a tumor suppressor. The SOD2 gene contains a large CpG island spanning > 3.5 kb that starts near the 5' edge of the promoter and extends into intron 2. We hypothesized that the difference in SOD2 expression between tumor cells and their normal cell counterparts might be secondary to differences in their cytosine methylation patterns in this CpG island. To test this hypothesis, we analyzed the methylation status of the SOD2 gene in two cell line models that show differential MnSOD expression between normal and SV40-transformed cells: WI38 and MRC5 and their SV40-transformed variants, WI38-VA and MRC5-VA. We subdivided the SOD2 gene CpG island into 10 individual regions for analysis by bisulfite genomic sequencing. A region located in intron 2 displayed a significant increase in cytosine methylation in both transformed cell lines that expressed low levels of MnSOD mRNA compared with their normal cell counterparts. Recent studies by others have shown that SOD2 intron 2 is a potent transcriptional enhancer. The association between increased cytosine methylation of the SOD2 intron 2 region and decreased MnSOD expression in transformed cells compared with their normal counterparts suggests that an epigenetic mechanism contributes to the differential SOD2 gene expression between these normal and SV40-transformed cells.

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We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning–based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the optical absorption spectra do not correlate with the optoacoustic spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

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