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.
Cancer cell invasion is crucial for the progression to metastatic disease, particularly, of cancers from epithelial origin, carcinomas, such as breast cancer. During the transition from a non-invasive to an invasive state, cancer cells exhibit many changes in cell shape and behavior. Characterizing these changes is essential for understanding the cellular properties of invasive cancer cells, and could provide novel therapeutic strategies targeting metastasis and/or novel biomarkers that predict the potential progression to metastasis, which remains to date the cause for the high mortality rate in cancer patients. In a paper recently published in the November issue of Cancer Cell, Gus Mouneimne from the Brugge lab and collaborators characterized a cellular process that controls invasive behavior. They established that the central player in this process, which regulates the cancer cells’ cytoskeleton is a biomarker for good prognosis in breast cancer patients, and that low expression of this actin regulator correlates with high risk of metastasis. This study could have clinical implications for the management of cancer progression, especially of high-grade tumors, which are often metastatic.
This cellular process that regulates invasion involves remodeling of the actin cytoskeletal architecture. In eukaryotic cells, the organization of the actin cytoskeleton influences cell shape and behavior. In this study, the Brugge lab describes a novel mechanism of actin cytoskeletal remodeling and propose a model explaining how altering the organization of the actin cytoskeleton affects cellular shape and behavior and promotes invasion. This actin remodeling process involves three main regulators: profilin-2, an actin binding protein that brings actin monomers to polymerization sites; EVL, an actin polymerization regulator of the Ena/VASP family of proteins; and myosin, a motor protein that promotes bundling and contractility of actin filaments. This work contributes to the advancement of understanding one of the initial steps of cancer metastasis, invasion into the tumor microenvironment.
Rodents use olfactory cues for species-specific behaviors. For example, mice emit odors to attract mates of the same species but not competitors of closely related species. This implies rapid evolution of olfactory signaling, although odors and chemosensory receptors involved are unknown. In recently published paper, the Liberles Lab identified a mouse chemosignal, trimethylamine, and its olfactory receptor, trace amine-associated receptor 5 (TAAR5), to be involved in species-specific social communication (Li et al, Current Biology, 2013). Abundant (>1,000-fold increased) and sex-dependent trimethylamine production arose de novo along the Mus lineage after divergence from Mus caroli. The two-step trimethylamine biosynthesis pathway involves synergy between commensal microflora and a sex-dependent liver enzyme, flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine. One key evolutionary alteration in this pathway is the recent acquisition in Mus of male-specific Fmo3 gene repression. Coincident with its evolving biosynthesis, trimethylamine evokes species-specific behaviors, attracting mice but repelling rats. Attraction to trimethylamine is abolished in TAAR5 knockout mice, and furthermore, attraction to mouse scent is impaired by enzymatic depletion of trimethylamine or TAAR5 knockout. TAAR5 is an evolutionarily conserved olfactory receptor required for a species-specific behavior. Synchronized changes in odor biosynthesis pathways and odor-evoked behaviors could ensure species-appropriate social interactions.
The cellular microenvironment can affect cell behavior not only through paracrine and autocrine factors that bind to cellular receptors, but also through the mechanical properties of the tissue. For example, it is known that the mechanical properties of the cell matrix, such as stiffness, can regulate the motility of single cells. However, it is not well understood how matrix stiffness affects collective migration, or the movement of groups of cells maintained by cell-cell adhesions. Collective migration plays critical roles in development, wound healing and cancer metastasis. To elucidate whether and how microenvironmental stiffness regulates collective migration, graduate student Rosa Ng (Brugge Lab) and postdoc Achim Besser (Danuser Lab) joined forces and conducted a systematic and quantitative study of epithelial sheet migration (Ng et al, Journal of Cell Biology, 2012).
Using a modified wound-healing assay, time-lapse microscopy and a custom cell migration tracking program, the migratory behaviors of >0.5 million MCF10A epithelial cells were monitored on substrates of various compliances. Individual cells comprising the epithelial sheets migrated faster, more persistently and more directionally on stiffer substrates. Most strikingly, increasing substrate stiffness promoted the coordination in cell movements during collective migration. On stiffer substrates, cell-cell coordination extended deeper from the wound edge into the cell sheet. Such propagation of cell-cell coordination was further correlated with myosin-II activity, in a manner dependent on cadherin-mediated cell-cell adhesions. Together, the results led to a physical model of collective migration, in which cell movements are regulated by microenvironmental stiffness modulation of cell contractility, and the coupling of cell contractility between cells by cadherin adhesions give rise to coordinated movements. The Brugge and Danuser laboratories are continuing to investigate the regulation of cell motility by the interplay between microenvironmental properties, cell contractility and cell-cell adhesions, which will be important for understanding developmental processes and cancer cell metastasis.