Michael Boyce

A decade of caspases.

Caspases are a family of cysteine proteases that play important roles in regulating apoptosis. A decade of research has generated a wealth of information on the signal transduction pathways mediated by caspases, the distinct functions of individual caspases and the mechanisms by which caspases mediate apoptosis and a variety of physiological and pathological processes.

Cellular response to endoplasmic reticulum stress: a matter of life or death.

The proper functioning of the endoplasmic reticulum (ER) is critical for numerous aspects of cell physiology. Accordingly, all eukaryotes react rapidly to ER dysfunction through a set of adaptive pathways known collectively as the ER stress response (ESR). Normally, this suite of responses succeeds in restoring ER homeostasis. However, in metazoans, persistent or intense ER stress can also trigger programmed cell death, or apoptosis. ER stress and the apoptotic program coupled to it have been implicated in many important pathologies but the regulation and execution of ER stress-induced apoptosis in mammals remain incompletely understood. Here, we review what is known about the ESR in both yeast and mammals, and highlight recent findings on the mechanism and pathophysiological importance of ER stress-induced apoptosis.

Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway

Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

Bringing chemistry to life

Bioorthogonal chemistry allows a wide variety of biomolecules to be specifically labeled and probed in living cells and whole organisms. Here we discuss the history of bioorthogonal reactions and some of the most interesting and important advances in the field.

Caspases: an ancient cellular sword of Damocles

AbstractCaspases are a family of cysteine proteases homologous to the Caenorhabditis elegans programmed cell death gene product CED-3. Caspases and their distant relatives, meta- and paracaspases, have been found in phylogenetically distant nonmetazoan groups, including plants, fungi and prokaryotes. This review summarizes the current information on the mechanisms and functions of non-mammalian caspases and their relatives in apoptotic and nonapoptotic processes, and explores the possible evolutionary origin of the caspase family.

A Chemical Method for Labeling Lysine Methyltransferase Substrates

Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin‐dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome‐wide identification of novel PKMT substrates. Our strategy makes use of an alkyne‐bearing S‐adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide‐functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof‐of‐concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

The channel of death

The proapoptotic members of the Bcl-2 family have been proposed to participate in the formation of a channel that releases these apoptogenic factors when mitochondria receive apoptotic signals. A recent study provides the first direct, biophysical measurement of a potentially apoptosis-specific mitochondrial channel, which is regulated by Bcl-2 family members and may play a primary role in the release of the proapoptotic factors.

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stress-induced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology.

A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses

The phosphorylation of the subunit α of eukaryotic translation initiation factor 2 (eIF2α), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2α remain unknown. Here, we provide evidence that eIF2α phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2α, additional pathways relying partly on Ca2+ flux may control eEF-2K activity during ER stress, as eIF2α phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context.