The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

AbstractThe heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFα-receptor activation as well as staurosporine treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase-3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis. Furthermore, we found that the anti-aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis

V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element

The immunoglobulin heavy chain intron enhancer (Eμ) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Eμ, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Eμ, the Eμ core as well as its flanking 5′ and 3′ matrix associated regions (5′ and 3′ MARs) upregulate V(D)J recombination while the downstream section of the 3′ MAR of Eμ does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3′ MAR of Eμ stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is not mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5′ and 3′ MARs of Eµ and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Eµ cor

Skin Calcium-Binding Protein Is a Parvalbumin of the Panniculus Carnosus

Skin calcium-binding protein (SCaBP) is a calcium binding protein purified from whole rat skin. It has a molecular weight of approximately 12,000 daltons but migrates at Mr 13,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. On nitrocellulose blots of SDS-polyacrylamide gels, 6 different antisera to SCaBP reacted equally well with SCaBP and parvalbumin (PV), an 11,500-dalton calcium-binding protein purified from rat skeletal muscle, which also migrates at Mr 13,000 on SDS-polyacrylamide gels. Rabbit antiserum to muscle PV also recognized both PV and SCaBP, and either protein absorbed specific antibodies against either antigen from both types of antisera. Soluble protein extracts from whole adult rat and mouse skin contained a Mr 13,000 protein which was recognized on nitrocellulose blots of SDS gels by both antisera. Blots of extracts from epidermis, dermis, whole skin, and skin scraped on the dermal side to remove hypodermal tissue revealed that the Mr 13,000 PV/SCaBP cross-reacting antigen was restricted to the hypodermal tissue removed by scraping. Immunofluorescent staining of Bouin-fixed skin sections with these antisera confirmed the localization of PV/SCaBP to the panniculus carnosus, a hypodermal muscle layer. Newborn mouse skin does not contain this antigen. Additional polypeptides of Mr 10,500 and 12,000 on SDS gels of extracts from the epidermis of newborn and adult rats and mice were found to be immunoreactive with anti-SCaBp serum. These polypeptides were not recognized by the PV antiserum, and the reactivity of anti-SCaBP for these antigens was not absorbed by purified PV or SCaBP. Our results indicate that SCaBP is antigenically indistinguishable from PV and is localized in the adult rodent panniculus carnosus, and that antisera to SCaBP are poly-specific, recognizing epidermal proteins in addition to SCaBP/PV

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Mice Null for Calsequestrin 1 Exhibit Deficits in Functional Performance and Sarcoplasmic Reticulum Calcium Handling

In skeletal muscle, the release of calcium (Ca2+) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca2+ release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca2+ buffering as well as its potential for modulating RyR1, the L-type Ca2+ channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca2+]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca2+ content and SR Ca2+ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca2+ release with single action potentials and a collapse of the Ca2+ release with repetitive trains. Under voltage clamp, SR Ca2+ release flux and total SR Ca2+ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca2+ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca2+ release, whereas the rapidly decaying component of SR Ca2+ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca2+] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca2+]free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca2+ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca2+ release

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The many faces of calmodulin in cell proliferation, programmed cell death, autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca2+ receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed. © 2013 Elsevier B.V.The research at the authors' labs was respectively funded by grants to MWB from the Danish Research Council, the Lundbeeck Foundation, AP Møller Fonden, Dagmar Marshall Fonden, Willumsen Fonden, Danielsen Fonden, Wedell Wedellsborg Fonden, Frænkels Mindefond, and Hansen Fonden; and grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494), the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), and the European Commission (contract PITN-GA-2011-289033).Peer Reviewe

Calmodulin protects cells from death under normal growth conditions and mitogenic starvation but plays a mediating role in cell death upon B-cell receptor stimulation

Calmodulin (CaM) is the main intracellular Ca(2+) sensor protein responsible for mediating Ca(2+) triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII(−/−) cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca(2+) levels remain elevated after the initial transient in CaMII(−/−) cells. Despite higher Ca(2+) resting levels, the CaMII(−/−) cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes