Katiuscia Bianchi

Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca2+, and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca2+-release channel inositol 1,4,5-trisphosphate receptor (IP3R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP3R on the ER or mitochondrial surface, which directly enhanced Ca2+ accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP3R and the mitochondrial Ca2+ uptake machinery. Because organelle Ca2+ homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

High glucose induces adipogenic differentiation of muscle-derived stem cells.

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria–localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death–inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.

A Tangled Web of Ubiquitin Chains: Breaking News in TNF-R1 Signaling

A flurry of recent revelations is challenging the current dogma on how ubiquitin-dependent processes culminate in the activation of NF-kappaB by TNF. Here, we integrate these findings into a model for TNF-R1 signaling-and underscore the importance of individual components, including linear ubiquitin chains-which allows for the remarkable versatility of the ubiquitin system.

Molecular determinants of Smac mimetic induced degradation of cIAP1 and cIAP2.

The inhibitors of apoptosis (IAP) proteins cIAP1 and cIAP2 have recently emerged as key ubiquitin-E3 ligases regulating innate immunity and cell survival. Much of our knowledge of these IAPs stems from studies using pharmacological inhibitors of IAPs, dubbed Smac mimetics (SMs). Although SMs stimulate auto-ubiquitylation and degradation of cIAPs, little is known about the molecular determinants through which SMs activate the E3 activities of cIAPs. In this study, we find that SM-induced rapid degradation of cIAPs requires binding to tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2). Moreover, our data reveal an unexpected difference between cIAP1 and cIAP2. Although SM-induced degradation of cIAP1 does not require cIAP2, degradation of cIAP2 critically depends on the presence of cIAP1. In addition, degradation of cIAP2 also requires the ability of the cIAP2 RING finger to dimerise and to bind to E2s. This has important implications because SM-mediated degradation of cIAP1 causes non-canonical activation of NF-κB, which results in the induction of cIAP2 gene expression. In the absence of cIAP1, de novo synthesised cIAP2 is resistant to the SM and suppresses TNFα killing. Furthermore, the cIAP2-MALT1 oncogene, which lacks cIAP2s RING, is resistant to SM treatment. The identification of mechanisms through which cancer cells resist SM treatment will help to improve combination therapies aimed at enhancing treatment response.

Mitochondrial calcium signalling in cell death

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.

Synchronous intra-Golgi transport induces the release of Ca2+ from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca(2+)) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca(2+) concentrations in the cell cytosol ([Ca(2+)](cyt)) and inside the lumen of the Golgi apparatus ([Ca(2+)](GA)), we have revealed transient increases in [Ca(2+)](cyt) during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca(2+)](GA) restoration ability. Thus, this redistribution of Ca(2+) from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca(2+)-dependent phase of SNARE-regulated fusion of Golgi compartments.

Mitochondrial calcium signalling in cell death.

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.

Ubiquitin-mediated regulation of RhoGTPase signalling: IAPs and HACE1 enter the fray

The EMBO Journal, 14–28 (2012); published online November252011 [PMC free article] [PubMed] Activation of members of the Rho-like family of guanosine triphosphatases GTPases (RhoGTPases) controls diverse physiological processes and is frequently found in cancer, contributing to tumour malignancy, cancer cell migration, invasion and metastasis. While the regulation of nucleotide binding to RhoGTPases is well understood, little is currently known regarding the molecular mechanisms through which RhoGTPase signalling is regulated by ubiquitylation. Two reports in this issue of The EMBO Journal and Developmental Cell now identify inhibitor of apoptosis (IAP) proteins and HACE1 as E3 ubiquitin (Ub)-protein ligases for Rac1 regulating Rac1 levels and activity. Most members of the RhoGTPase protein family function as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound state (Vega and Ridley, 2008). Binding of GTP to RhoGTPases, such as Rac1, triggers a conformational change that allows binding and activation of downstream effector proteins, through which Rac1 modulates actin assembly, actomyosin contractility, and microtubule formation. Activation of RhoGTPases, and hence their potential to interact with downstream signalling molecules, is influenced by a range of auxiliary proteins. Guanine-nucleotide exchange factors (GEFs) catalyse the exchange of GDP for GTP, thus activating the RhoGTPase. By contrast, GTPase-activating proteins (GAPs) promote the intrinsic GTPase activity, thereby accelerating the hydrolysis of GTP to GDP and returning RhoGTPases to their inactive configuration. RhoGTPases can also be kept inactive by association with cytosolic chaperone proteins known as guanine-nucleotide dissociation inhibitors (GDIs), which block the action of GEFs, and extract GDP-bound RhoGTPases from membranes where they may otherwise become activated inappropriately. In addition to modulation by GEFs, GAPs and GDIs, RhoGTPases are subject to regulation via post-translational modification. For example, addition of lipidic groups to their C-termini enhance their interaction with membranes, while conjugation of Ub to RhoGTPases targets them for inactivation via proteasomal degradation (Nethe and Hordijk, 2010). By interrogating the role of IAP proteins in health and disease, Krishnaraj Rajalingam and colleagues noticed that silencing of the E3s XIAP and cIAP1 led to Rac1 stabilisation, elongated morphology and enhanced migration (Oberoi et al, 2012) (see Figure 1). Consistent with this notion, they demonstrate that XIAP and cIAP1 directly bind to Rac1 in a nucleotide-independent manner in vitro and in vivo, and that both these IAPs can conjugate polyubiquitin chains to Rac1, targeting it for proteasomal degradation. They further demonstrate that these IAPs are also required for Rac1 degradation following expression of RhoGDI, and exposure to cytotoxic necrosis factor 1 (CNSF1)—a toxin that is produced by urophathogenic Escherichia coli (UPEC) and targets members of the RhoGTPases, including Rac1, for proteasomal degradation. In the absence of XIAP and cIAPs, however, RhoGDI- and CNSF1-mediated depletion of Rac1 is prevented. IAPs appear not to be the only E3s that target Rac1 for proteasomal degradation following CNSF1 intoxication. Elegant work by Emmanuel Lemichez and colleagues demonstrate that the HECT-type E3 HACE1 is required for CNSF1-mediated depletion of Rac1 (Torrino et al, 2011) (see Figure 1). They further demonstrate that in the absence of HACE1, UPEC fail to efficiently invade endothelial cell monolayers, suggesting that HACE1 plays a major role in host defence against pathogens. The relative contributions of IAPs and HACE1 to Rac1 turnover remain to be clarified, but it is interesting to note that while HACE interacts specifically with active Rac1, IAPs seem to be indiscriminate about the activation state of Rac1. Figure 1 Schematic overviews of Ub-mediated regulation of Rac1. The activity status of Rac1 is determined by its association with GAPs, GEFs and GDIs as well as through post-translational modifications. Ubiquitylation ... IAPs also regulate migration of certain neuronal progenitor cells of the developing cerebellum of zebrafish. Normally, cells migrate from the upper rhombic lip region of the cerebellum to their final destination in several of the tegmental hindbrain nuclei and in the cerebellar cortex (Tahirovic et al, 2010). Strikingly, either expression of a dominant-negative form of Rac1, or overexpression of XIAP, in the upper rhombic lip cells impaired directional migration of these cells to their final destination. The phenotype caused by XIAP overexpression was partially rescued by co-expression of wild-type Rac1, indicating that XIAP exerts its effects through Rac1. The ability of mammalian IAPs to regulate migration via controlling Rac1 appears to be evolutionarily conserved, as the Drosophila IAP1 (DIAP1) has previously been reported to bind to Rac and regulate collective migration of a group of cells in the developing egg chamber (Geisbrecht and Montell, 2004). However, while the report by Oberoi et al, and previous data from the same group (Dogan et al, 2008), suggest that IAPs inhibit cell migration, the data from Drosophila (Geisbrecht and Montell, 2004), and other reports from mammalian systems (Mehrotra et al, 2010; Liu et al, 2011; Lopez et al, 2011), indicate that IAPs actually promote migration. Clarification of this discrepancy will be extremely important, not least because pharmacological inhibitors of IAPs (dubbed Smac mimetics) are undergoing clinical trials for the treatment of cancer (Dynek and Vucic, 2010). While Smac mimetics were developed to inactivate IAPs and induce tumour cell death, the study by Rajalingam and colleagues now suggests that treatment with IAP inhibitors may promote migration of surviving tumour cells and thus increase the risk of metastasis. Unravelling how IAPs regulate cell migration and invasion will be critically important, as metastasis represents the major clinical problem in cancer.

Aerobic glycolysis: beyond proliferation.

Aerobic glycolysis has been generally associated with cancer cell proliferation, but fascinating and novel data show that it is also coupled to a series of further cellular functions. In this Mini Review, we will discuss some recent findings to illustrate newly defined roles for this process, in particular in non-malignant cells, supporting the idea that metabolism can be considered as an integral part of cellular signaling. Consequently, metabolism should be regarded as a plastic and highly dynamic determinant of a wide range of cellular specific functions.