Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.

Death is a vital developmental cell fate. In Caenorhabditis elegans, programmed death of the linker cell, which leads gonadal elongation, proceeds independently of caspases and apoptotic effectors. To identify genes promoting linker-cell death, we performed a genome-wide RNA interference screen. We show that linker-cell death requires the gene pqn-41, encoding an endogenous polyglutamine-repeat protein. pqn-41 functions cell autonomously, and is expressed at the onset of linker-cell death. pqn-41 expression is controlled by the MAP kinase kinase SEK-1, which functions in parallel to the Zn-finger protein LIN-29 to promote cellular demise. Linker-cell death is morphologically similar to cell death associated with normal vertebrate development and polyglutamine-induced neurodegeneration. Our results may, therefore, provide molecular in-roads to understanding non-apoptotic cell death in metazoan development and disease.

Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.DOI: http://dx.doi.org/10.7554/eLife.12821.001

Summary Polyglutamine-repeat diseases are neurodegenerative ailments elicited by glutamine-encoding CAG nucleotide expansions within endogenous human genes. Despite efforts to understand the basis of these diseases, the precise mechanism of cell death remains stubbornly unclear. Much of the data seems consistent with a model in which toxicity is an inherent property of the polyglutamine repeat, whereas host protein sequences surrounding the polyQ expansion modulate severity, age of onset, and cell specificity. Recently, a gene, pqn-41encoding a glutamine-rich protein was found to promote normally-occurring non-apoptotic cell death in C. elegans. Here we review evidence for toxic and modulatory roles for polyQ repeats and their host proteins, respectively, and suggest similarities with pqn-41 function. We explore the hypothesis that toxicity mediated by glutamine-rich motifs may be important not only in pathology, but also in normal development.

The temporal and spatial control of protein function is of fundamental importance in biology. Most cellular processes require that a small subset of the proteome be active at a particular time and place, and that this activity have a defined duration. To probe the role of a protein in a biological system, one must be able to control these parameters as precisely as possible. [1,2] We recently developed a new tool, termed conditional protein splicing (CPS), to control the primary structure, and hence function, potentially of any protein by using a small molecule. [3,4] The basic principle of CPS is illustrated in Figure 1 a. Exploitation of a split intein that is only active in the presence of the small molecule rapamycin allows proteins or polypeptides to be linked by a peptide bond through protein splicing. The sequences of interest are expressed as recombinant fusions to the N-and C-terminal halves of the intein, which are themselves linked to FKBP and FRB domains. These domains form a high-affinity ternary complex with rapamycin.[5] The induced proximity of the intein halves in this complex mediates the reconstitution of the active intein. Since CPS acts at the posttranslational level, it has the advantage of a short response time (as little as 10 min), which allows high temporal resolution.There are many conceivable strategies for specifically altering the function of a protein by CPS. The most obvious method is to reassemble the protein from two inactive pieces and thereby switch on its activity. This strategy exploits the bond-making feature of CPS and requires that the newly spliced polypeptide spontaneously adopts an active structure. We decided to take advantage of another feature of the CPS reaction, the peptide-bond-breaking steps. The CPS system depicted in Figure 1 a can be regarded not only as a conditional protein ligase that forms the peptide bond between the two extein sequences, but also as a conditional protease that breaks the peptide bonds between the intein and extein sequences. We conceived a strategy in which the protein of interest and a peptide sequence that acts as an inhibitor are fused to opposite ends of one of the CPS constructs such that they are cleaved from one another in the course of the protein splicing reaction. This cleavage should result in a relative increase in the activity of the protein because the inhibitor should display a lower potency when free than while fused to the protein as a result of its higher local concentration in the intramolecular arrangement. This design borrows from the principles often used by nature to control the activity of enzymes. So-called active-site-directed intrasteric autoregulation [6] has been observed for many proteins, for example, zymogens are kept in an autoinhibited state until posttranslational processing reveals the active protease.Protein kinases are key players in a myriad of important processes in the cell and are thus attractive targets upon which to test our idea. We chose the cAMP-dependent protein kinase (PKA) for our investi...