Cellular stress management by caspases.

Cellular stress plays a pivotal role in the onset of numerous human diseases. Consequently, the removal of dysfunctional cells, which undergo excessive stress-induced damage via various cell death pathways, including apoptosis, is essential for maintaining organ integrity and function. The evolutionarily conserved family of cysteine-aspartic-proteases, known as caspases, has been a key player in orchestrating apoptosis. However, recent research has unveiled the capability of these enzymes to govern fundamental cellular processes without triggering cell death. Remarkably, some of these non-lethal functions of caspases may contribute to restoring cellular equilibrium in stressed cells. This manuscript discusses how caspases can function as cellular stress managers and their potential impact on human health and disease. Additionally, it sheds light on the limitations of caspase-based therapies, given our still incomplete understanding of the biology of these enzymes, particularly in non-apoptotic contexts.

A LexAop > UAS > QUAS trimeric plasmid to generate inducible and interconvertible Drosophila overexpression transgenes.

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAS) has greatly expanded versatile genetic analyses in the Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of noninterchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAS modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

The LMTK-family of kinases: Emerging important players in cell physiology and pathogenesis.

Lemur Tail (former tyrosine) Kinases (LMTKs) comprise a novel family of regulated serine/threonine specific kinases with three structurally and evolutionary related members. LMTKs exercise a confusing variety of cytosolic functions in cell signalling and membrane trafficking. Moreover, LMTK2 and LMTK3 also reside in the nucleus where they participate in gene transcription/regulation. As a consequence, LMTKs impact cell proliferation and apoptosis, cell growth and differentiation, as well as cell migration. All these fundamental cell behaviours can turn awry, most prominently during neuropathologies and tumour biogenesis. In cancer cells, LMTK levels are often correlated with poor overall prognosis and therapy outcome, not least owned to acquired drug resistance. In brain tissue, LMTKs are highly expressed and have been linked to neuronal and glia cell differentiation and cell homeostasis. For one member of the LMTK-family (LMTK2) a role in cystic fibrosis has been identified. Due to their role in fundamental cell processes, altered LMTK physiology may also warrant a hitherto unappreciated role in other diseases, and expose them as potential valuable drug targets. On the backdrop of a compendium of LMTK cell functions, we hypothesize that the primary role of LMTKs may dwell within the endocytic cargo recycling and/or nuclear receptor transport pathways.

Non-apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence.

Caspase malfunction in stem cells often precedes the appearance and progression of multiple types of cancer, including human colorectal cancer. However, the caspase-dependent regulation of intestinal stem cell properties remains poorly understood. Here, we demonstrate that Dronc, the Drosophila ortholog of caspase-9/2 in mammals, limits the number of intestinal progenitor cells and their entry into the enterocyte differentiation programme. Strikingly, these unexpected roles for Dronc are non-apoptotic and have been uncovered under experimental conditions without epithelial replenishment. Supporting the non-apoptotic nature of these functions, we show that they require the enzymatic activity of Dronc, but are largely independent of the apoptotic pathway. Alternatively, our genetic and functional data suggest that they are linked to the caspase-mediated regulation of Notch signalling. Our findings provide novel insights into the non-apoptotic, caspase-dependent modulation of stem cell properties that could improve our understanding of the origin of intestinal malignancies.

Shedding of bevacizumab in tumour cells-derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma.

Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity.

LMTK3 confers chemo-resistance in breast cancer.

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.

Extracellular vesicles round off communication in the nervous system.

Functional neural competence and integrity require interactive exchanges among sensory and motor neurons, interneurons and glial cells. Recent studies have attributed some of the tasks needed for these exchanges to extracellular vesicles (such as exosomes and microvesicles), which are most prominently involved in shuttling reciprocal signals between myelinating glia and neurons, thus promoting neuronal survival, the immune response mediated by microglia, and synapse assembly and plasticity. Such vesicles have also been identified as important factors in the spread of neurodegenerative disorders and brain cancer. These extracellular vesicle functions add a previously unrecognized level of complexity to transcellular interactions within the nervous system.

Tumor-Stromal Cell Communication: Small Vesicles Signal Big Changes.

Reciprocal interactions between malignant and stromal cells create a local microenvironment that fosters tumor growth. Extracellular vesicles (EVs) such as exosomes, microvesicles, and large oncosomes are involved in tumor-stroma communication by shuttling signaling cargo and other molecules. Here we discuss how EVs released by cancer or stromal cells impact the proliferation, differentiation, and metabolism of tumors.

The ESCRT machinery regulates the secretion and long-range activity of Hedgehog.

The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

Cancer becomes wasteful: emerging roles of exosomes(†) in cell-fate determination.

Extracellular vesicles (EVs), including exosomes, have been widely recognized for their role in intercellular communication of the immune response system. In the past few years, significance has been given to exosomes in the induction and modulation of cell-fate-inducing signalling pathways, such as the Hedgehog (Hh), Wnts, Notch, transforming growth factor (TGF-ß), epidermal growth factor (EGF) and fibroblast growth factor (FGF) pathways, placing them in the wider context of development and also of cancer. These protein families induce signalling cascades responsible for tissue specification, homeostasis and maintenance. Exosomes contribute to cell-fate signal secretion, and vice versa exosome secretion can be induced by these proteins. Interestingly, exosomes can also transfer their mRNA to host cells or modulate the signalling pathways directly by the removal of downstream effector molecules from the cell. Surprisingly, much of what we know about the function of exosomes in cell determination is gathered from pathological transformed cancer cells and wound healing while data about their biogenesis and biology in normal developing and adult tissue lag behind. In this report, we will summarize some of the published literature and point to current advances and questions in this fast-developing topic. In a brief foray, we will also update and shortly discuss their potential in diagnosis and targeted cancer treatment.

A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway.

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.

Disruption of Vps4 and JNK function in Drosophila causes tumour growth.

Several regulators of endocytic trafficking have recently been identified as tumour suppressors in Drosophila. These include components of the endosomal sorting complex required for transport (ESCRT) machinery. Disruption of subunits of ESCRT-I and -II leads to cell-autonomous endosomal accumulation of ubiquitinated receptors, loss of apicobasal polarity and epithelial integrity, and increased cell death. Here we report that disruption of the ATPase dVps4, the most downstream component of the ESCRT machinery, causes the same array of cellular phenotypes. We find that loss of epithelial integrity and increased apoptosis, but not loss of cell polarity, require the activation of JNK signalling. Abrogation of JNK signalling prevents apoptosis in dVps4 deficient cells. Indeed double deficiency in dVps4 and JNK signalling leads to the formation of neoplastic tumours. We conclude that dvps4 is a tumour suppressor in Drosophila and that JNK is central to the cell-autonomous phenotypes of ESCRT-deficient cells.

In vivo role of lipid adducts on Wingless.

Two lipids (palmitate and palmitoleic acid) are appended onto Wnt proteins. It has been suggested that palmitate is required for signalling, whereas palmitoleic acid is necessary for progression through the secretory pathway. By mutating the relevant amino acids, we have investigated how these adducts contribute to the secretion and signalling activity of Wingless, the main Drosophila member of the Wnt family. Analysis of Wingless with a Cysteine 93 to Alanine mutation ([C93A]Wingless) shows that palmitoylation is essential for signalling activity in vivo (as well as in cultured cells). Moreover, without palmitate, Wingless fails to reach the surface of imaginal disc cells and, as electron microscopy (EM) analysis suggests, appears to accumulate in the endoplasmic reticulum (ER). Artificial targeting of palmitate-deficient Wingless to the plasma membrane does not rescue signalling activity. Therefore, palmitate at C93 has a dual role: in secretion and signalling. From our analysis of [S239A]Wingless, which lacks a conserved residue shown to be acylated in Wnt3a, we infer that palmitoleic acid is not, as previously suggested, absolutely required for secretion. Nevertheless, this mutant has poor signalling activity, suggesting that palmitoleic acid contributes significantly to signalling. We suggest that the overall level of lipidation affects signalling activity.

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex.

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.

ESCRTs and Fab1 regulate distinct steps of autophagy.

Eukaryotes use autophagy to turn over organelles, protein aggregates, and cytoplasmic constituents. The impairment of autophagy causes developmental defects, starvation sensitivity, the accumulation of protein aggregates, neuronal degradation, and cell death [1, 2]. Double-membraned autophagosomes sequester cytoplasm and fuse with endosomes or lysosomes in higher eukaryotes [3], but the importance of the endocytic pathway for autophagy and associated disease is not known. Here, we show that regulators of endosomal biogenesis and functions play a critical role in autophagy in Drosophila melanogaster. Genetic and ultrastructural analysis showed that subunits of endosomal sorting complex required for transport (ESCRT)-I, -II and -III, as well as their regulatory ATPase Vps4 and the endosomal PtdIns(3)P 5-kinase Fab1, all are required for autophagy. Although the loss of ESCRT or Vps4 function caused the accumulation of autophagosomes, probably because of inhibited fusion with the endolysosomal system, Fab1 activity was necessary for the maturation of autolysosomes. Importantly, reduced ESCRT functions aggravated polyglutamine-induced neurotoxicity in a model for Huntington's disease. Thus, this study links ESCRT function with autophagy and aggregate-induced neurodegeneration, thereby providing a plausible explanation for the fact that ESCRT mutations are involved in inherited neurodegenerative disease in humans [4].

How does cholesterol affect the way Hedgehog works?

Members of the Hedgehog (Hh) family of proteins are conserved morphogens that spread and modulate cell fates in target tissue. Mature Hh carries two lipid adducts, a palmitoyl group at the N terminus and cholesterol at the C terminus. Recent findings have addressed how these lipid modifications affect the function and transport of Hh in Drosophila. In contrast to the palmitoyl adduct, cholesterol appears not to be essential for signalling. However, the absence of the cholesterol adduct affects the spread of Hh within tissues. As we discuss here, the exact nature of this effect is controversial.

Functional genomics reveals genes involved in protein secretion and Golgi organization.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

AP-1 recruitment to VAMP4 is modulated by phosphorylation-dependent binding of PACS-1.

The R-SNARE VAMP4, which contains a dileucine motif, binds to the AP-1 (adaptor protein-1) subunit mu 1a, but not mu 1b, or the GGAs (Golgi-associated gamma ear containing ARF binding proteins). Serine 20 and leucines 25,26 are essential for this binding. AP-1 association with VAMP4 is enhanced when serine 30, in an acidic cluster, is phosphorylated by casein kinase 2. This phosphorylation-dependent modulation of AP-1 binding is mediated by PACS-1 (phosphofurin acidic cluster sorting protein). Ablation of both the dileucine motif and serine 30 results in a dramatic mislocalization of VAMP4 in the regulated secretory pathway in AtT20 cells. A dominant-negative PACS-1, which binds acidic clusters but not AP-1, also causes mislocalization of VAMP4. Our data support a model whereby phosphorylation-dependent recruitment of PACS-1 enhances AP-1 association to cargo, and suggest that efficient retrieval depends on the formation of a complex between cargo, such as VAMP4, AP-1 and PACS-1.

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization.

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.