Histones and histone-binding proteins play important role in activating and silencing eukaryotic genes. How binding of factors to the nucleosome may change the properties of chromatin to mediate gene activation or silencing is not understood. A recent crystal structure by Wang et al. (PNAS, 2013) of the nucleosome complexed with the BAH domain of the heterochromain protein Sir3 sheds new light on this question.
Heterochromatic gene silencing in the budding yeast S. cerevisiae requires a nucleosome binding protein, Sir3, and specific histone amino acids, in particular a conserved basic patch region spanning amino acids 16 to 20 in the N-terminal tail of histone H4. Wang and colleagues now provide evidence that association of Sir3 with the nucleosome induces interactions between the N-terminal tail of histone H4 and nucleosomal DNA. The Moazed Lab had previously shown that a conserved region in the N-terminus of Sir3, called the BAH domain, is a nucleosome- and histone tail-binding domain. In this paper, they show that while some histone H4 side chains are critical for binding of Sir3 to the nucleosome, two arginines (R17 and R19), which are critical for silencing in vivo, have no effect on binding of Sir3 to nucleosomes in vitro. To understand the paradoxical requirement for these arginines in gene silencing, the authors determined the 3.1 Å resolution crystal structure of the Sir3-BAH domain in complex with the nucleosome. Consistent with their biochemical data, the structure shows that R17 and R19 point away from the BAH domain and make electrostatic contacts with phosphates of the nucleosomal DNA backbone. In contrast, other histone side chains, which are also critical for silencing, make extensive bonding interactions with the BAH domain. These observations suggest a new role for histone tails in gene silencing and heterochromatin formation, beyond assembly platforms for recruitment of regulatory factors.
Cancer cells require the biosynthesis of structural components for biomass production. This requires a reprogramming of the metabolic pathways to ensure that nutrients such as glucose and glutamine are not completely oxidized but instead their intermediary metabolites are shunted into biosynthetic pathways. Activation of the mammalian Target of Rapamycin Complex 1 (mTORC1) has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutamine metabolism has been reported. A recent paper from the Blenis Lab (Csibi et al., Cell, 2013) showed that mTORC1 activation regulates glutamine anaplerosis by promoting the conversion of glutamine to α-ketoglutarate, a tricarboxylic acid (TCA) cycle intermediate. Csibi and colleagues found that mTORC1 promotes the activity of glutamate dehydrogenase (GDH). The authors sought to identify how this occurred and found that SIRT4, an inhibitor of GDH, was repressed by mTORC1. The molecular detail of this repression involved the mTORC1-mediated phosphorylation and proteasome-mediated degradation of CREB2, a transcription factor that binds the SIRT4 human promoter. The expression of SIRT4 mRNA was decreased in several human cancers, and its forced expression in cancer cells significantly repressed proliferation, and resulted in reduced tumor development in a TSC-xenograft model. These findings suggested that targeting glutamine metabolism might impair the growth or survival of cancer cells. Indeed, the authors found that the combined pharmacological inhibition of glucose and glutamine metabolism selectively sensitized mTORC1-active cells to undergo cell death. Altogether, these results point to a central role for mTORC1 in the regulation of glutamine metabolism and suggest that the use of glutamine metabolism inhibitors may prove efficacious in nutrient-dependent cancers with high mTORC1 signaling.
Genomic instability and altered metabolism are key features of many cancer cells. Thus, defining pathways involved in regulating DNA damage responses and cellular metabolism hold important implications for understanding normal cell growth, as well as for the development of strategies to prevent or treat cancer. A recent paper from the Haigis Lab (Jeong et al, Cancer Cell, 2013) sheds new light on understanding of how cells adapt their metabolism in response to DNA damage. Jeong and colleagues report that a mitochondria-localized sirtuin, SIRT4, is up-regulated in response to DNA-damaging agents, and coordinates repression of mitochondrial glutamine metabolism with cell arrest. Loss of SIRT4 promotes genomic instability and tumor growth in mice. Moreover, SIRT4 expression is decreased in many human cancers. These findings suggest that SIRT4 has tumor suppressive activity, and provides a new link between glutamine metabolism and genomic integrity.
The Notch signaling pathway links the fate of one cell to that of the next door cellular neighbor. The developmental action of Notch is pleiotropic and in general Notch activity is associated with early lineages in a very broad spectrum of tissues and organs. Consequently the Notch receptor is expressed and controls the fate of many early precursors and indeed stem cells. Based on this, the Artavanis-Tsakonas Lab developed a series of transgenic mice in which an inducible form of the Cre recombinase has been knocked in, respectively, the Notch 1,2,3 and 4 receptor genes, thus expressing Cre in cells expressing each receptor paralogue (Sale et al, Nat Cell Bio, 2013). Using these tools they have been determining cell lineages and in this study they followed cell lineages associated with Notch 2 in the mammary gland. They thus uncovered two new epithelial lineages that which allow them to revise the classical cellular hierarchies associated with mammary development. They thus revise current models of mammary epithelial cell hierarchy and reveal a hitherto undescribed mechanism regulating branching morphogenesis. These results may have implications for the identification of the cell-of-origin of distinct breast cancer subtypes.
The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy. While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM, the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome – remains poorly defined. Graduate student Shireen Sarraf (Harper Lab) and colleagues used a method called "quantitative diGLY proteomics" to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization (Sarraf et al, Nature, 2013). This method was developed previously by postdocs Woong Kim (Gygi Lab) and Eric Bennett (Harper Lab) (Molecular Cell, 2011). Sarraf et al. identified hundreds of dynamically regulated ubiquitylation sites in dozens of proteins, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, Sarraf et al. found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis, and can be interrogated via a website constructed by Mat Sowa (Harper Lab) that allows the dynamics and structural biology of modification sites to be mined. This paper provides the first global view of the site specificity for target modification by any E3 ubiquitin ligase.
Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-γ: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-γ treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, the Goldberg lab [Nathan JA, Spinnenhirn V, Schmidtke G, Basler M, Groettrup M, Goldberg AL. Immuno- and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins. Cell. 2013 Feb 28;152(5):1184-94] reports that polyubiquitin conjugates do not transiently accumulate following IFNγ-treatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles.
Tumor cells select and reprogram a variety of central metabolic and bioenergetic pathways to maintain exacerbated growth and survival rates. The identification of the genetic factors responsible for the execution of these specific metabolic programs is key to exploit this reprogramming for cancer therapy. A paper from the Puigserver Lab (Vazquez et al, Cancer Cell, 2013) shows that in melanomas, overexpression of the transcriptional coactivator PGC1α, a key regulator of mitochondrial respiration, metabolically defines melanoma tumors with high mitochondrial bioenergetic and ROS detoxification capacities. These reprogrammed metabolic capacities allow PGC1α-positive melanomas higher rates of survival under oxidative stress compared to PGC1α-negative melanomas. These findings underscore how different metabolic vulnerabilities defined by PGC1α expression could be therapeutically exploited to treat melanoma tumors.
An essential requirement for cell growth is the availability of sufficient nutrients to meet a cell’s biological demands. These nutrients include glucose and glutamine, both of which ultimately enter the TCA cycle, providing the cell with carbon sources to be used for synthesis of macromolecules as well as redox potential to generate ATP. A key regulator of cell growth is the mammalian target of rapamycin complex 1 (mTORC1), which senses the availability of nutrients in the environment, and drives cell growth and proliferation under nutrient sufficient conditions through coordination of anabolic/catabolic processes. Despite the necessity of glucose and glutamine for energy production and macromolecule synthesis, it is not fully understood how mTORC1 senses the availability of these two nutrients in order to properly coordinate cell growth. In a paper recently published in the January issue of Molecular Cell, the Blenis lab and colleagues find that glucose and glutamine, through production of ATP via the TCA cycle, positively feed back to mTORC1 activation through an AMPK-, TSC1/2-, and Rag-independent mechanism. Through completion of an siRNA screen, Blenis lab members identified the ATP-dependent Tel2-Tti1-Tti2 (TTT)-RUVBL1/2 complex as a metabolic regulator of mTORC1 signaling. This complex has previously been shown to regulate the assembly and stability of phoshphoinositide-3-kinase-related protein kinases (PIKK) family proteins, including mTORC1. In this study, the Blenis lab showed that energetic stress caused by the depletion of glucose and glutamine leads to disassembly and repression of the TTT-RUVBL1/2 complex, and that this inhibition prevents homodimerization of mTORC1 into a functional signaling complex and its lysosomal localization – processes that are necessary for phosphorylation of mTORC1 substrates. Moreover, expression of the TTT-RUVBL1/2 complex was found to be upregulated in several tumors and this upregulation was positively correlated with mTORC1 signaling. This study demonstrates novel roles of the TTT-RUVBL1/2 complex in sensing cellular metabolic state, and accordingly regulating cell growth by controlling mTORC1’s functional assembly and lysosomal localization. These findings further suggest that the TTT-RUVBL1/2 complex has potential roles in promoting tumorigenesis, in part through energy-dependent mTORC1 assembly and regulation of this critical cell growth pathway.