Protein Turnover in Brain of the Rat Fetus

Protein synthesis in vivo was studied in whole brain of rat fetuses using continuous intravenous infusion of L-[U-14C]tyrosine into unrestrained pregnant rats at 19 and 21 days gestation. Protein degradation (KD) was calculated by subtracting fractional growth rate of brain protein (KG) from the fractional synthesis rate (KS). KS was high at both gestational ages (0.42 +/- 0.03 days-1 at day 19, 0.47 +/- 0.029 days-1 at 21 days), comparable to values previously reported for newborn rat cerebral hemispheres, and threefold higher than is seen in adult animals. KD was similar at both 19 and 21 days gestation (0.19-0.24) and lower than that reported in neonatal rat brain using similar techniques. Protein accretion during the most rapid phase of brain growth (fetus) is accomplished by similar rates of protein synthesis, but decreased rates of degradation when compared with a slower growth phase (newborn). KD in the brain of the rapidly growing fetus is slightly higher than in adult cerebral hemispheres.     

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [³²P]-labeled photoreactive partial DNA duplexes containing a 3′-ss/ds-junction (3′-junction) or a 5′-ss/ds-junction (5′-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3′-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5′-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5′-junction. The results show that RPAp70 crosslinked to DNA with a 5′-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Snap,crackle, and pop: Acoustic-based model estimation of snapping shrimp populations in healthy and degraded hard-bottom habitats

Human use of the ocean and its ecosystems continues to degrade coastal habitats around the world. Assessing anthropogenic impacts on these environments can be costly and manpower-intensive; thus, the development of rapid, remote techniques to assess habitat quality is important. We employed autonomous hydrophone receivers to record the soundscapes of healthy, sponge-rich hard-bottom habitat in Florida Bay, Florida (USA) and hard-bottom areas impacted by sponge die-offs. We also recorded sounds emanating from individual sponges of three species that were isolated in underwater sound booths, and then enumerated the invertebrates (mostly snapping shrimp) dwelling within the canals of each sponge. From these recordings, a modified cylindrical sound propagation model was used to estimate distances to individual snapping shrimp snaps. Using the program Distance, we estimated snapping shrimp population density and abundance within both habitat types. More snapping shrimp snaps per unit time were recorded in healthy hard-bottom areas as compared to degraded hard-bottom areas. In addition, the average distance to a snap source was greater within degraded hard-bottom areas than within healthy hard-bottom areas. As a consequence, the estimated density and abundance of snapping shrimp were one to two orders of magnitude greater within healthy habitat than within degraded habitat. This study demonstrates the feasibility of using acoustic sampling and modeling to rapidly assess populations of soniferous benthic indicator species, whose vocalizations may yield indirect estimates of habitat quality.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).     

What is Killing Organic Photovoltaics: Light‐Induced Crosslinking as a General Degradation Pathway of Organic Conjugated Molecules

In view of a rapid development and increase in efficiency of organic solar cells, reaching their long‐term operational stability represents now one of the main challenges to be addressed on the way toward commercialization of this photovoltaic technology. However, intrinsic degradation pathways occurring in organic solar cells under realistic operational conditions remain poorly understood. The light‐induced dimerization of the fullerene‐based acceptor materials discovered recently is considered to be one of the main causes for burn‐in degradation of organic solar cells. In this work, it is shown that not only the fullerene derivatives but also different types of conjugated polymers and small molecules undergo similar light‐induced crosslinking regardless of their chemical composition and structure. In the case of conjugated polymers, crosslinking of macromolecules leads to a rapid increase in their molecular weight and consequent loss of solubility, which can be revealed in a straightforward way by gel permeation chromatography analysis via a reduction/loss of signal and/or smaller retention times. Results of this work, thus, shift the paradigm of research in the field toward designing a new generation of organic absorbers with enhanced intrinsic photochemical stability in order to reach practically useful operation lifetimes required for successful commercialization of organic photovoltaics.     

Investigating the Effects of Chemical Gradients on Performance and Reliability within Perovskite Solar Cells with TOF‐SIMS

Time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS), a powerful analytical technique sensitive to all components of perovskite solar cell (PSC) materials, can differentiate between the various organic species within a PSC absorber or a complete device stack. The ability to probe chemical gradients through the depth of a device (both organic and inorganic), with down to 100 nm lateral resolution, can lead to unique insights into the relationships between chemistry in the absorber bulk, at grain boundaries, and at interfaces as well as how they relate to changes in performance and/or stability. In this review, the technique is described; then, from the literature, several examples of how TOF‐SIMS have been used to provide unique insight into PSC absorbers and devices are covered. Finally, the common artifacts that can be introduced if the data are improperly collected, as well as methods to mitigate these artifacts are discussed.