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Legionella pneumophila, a Gram-negative intracellular bacterium, is one of the major causes of Legionnaires’ disease, a specific type of atypical pneumonia. Despite intensive research efforts that elucidated many relevant structural, molecular and medical insights into Legionella’s pathogenicity, Legionnaires’ disease continues to present an ongoing public health concern. Legionella’s virulence is based on its ability to simultaneously hijack multiple molecular pathways of the host cell to ensure its fast replication and dissemination. Legionella usurps the host ubiquitin system through multiple effector proteins, using the advantage of both conventional and unconventional (phosphoribosyl-linked) ubiquitination, thus providing optimal conditions for its replication. In this review, we summarize the current understanding of L. pneumophila from medical, biochemical and molecular perspectives. We describe the clinical disease presentation, its diagnostics and treatment, as well as host-pathogen interactions, with the emphasis on the ability of Legionella to target the host ubiquitin system upon infection. Furthermore, the interdisciplinary use of innovative technologies enables better insights into the pathogenesis of Legionnaires’ disease and provides new opportunities for its treatment and prevention.
Sorting nexins are a conserved protein family involved in vesicle transport, membrane trafficking and protein sorting. The sorting nexin ATG24/SNX4 has been demonstrated to be involved in different autophagy pathways and in endosomal trafficking. However, its impact on cellular quality control and on aging and development is still elusive. Here we report studies analyzing the function of PaATG24 in the aging model Podospora anserina. Ablation of PaATG24 leads to a reduced growth rate, infertility, and to a pronounced lifespan reduction. These characteristics are accompanied by alterations of the morphology and size distribution of vacuoles and severe impairments in non-selective and selective autophagy of peroxisomes (pexophagy) and mitochondria (mitophagy). While general autophagy and pexophagy are almost completely blocked, a PaATG24-independent form of mitophagy is induced during aging. In the ΔPaAtg24 mutant a strong accumulation of peroxisomes occurs while mitochondrial abundance is only slightly increased. These mitochondria are partially affected in function. Most strikingly, although some PaATG24-independent mitophagy exists, it appears that this is not sufficient to remove dysfunctional mitochondria efficiently enough to prevent premature aging. Overall our data emphasize the key role of mitochondria in aging and of mitophagy in quality control to keep a population of “healthy” mitochondria during aging.
Chaperone-assisted selective autophagy (CASA) initiated by the cochaperone Bcl2-associated athanogene 3 (BAG3) represents an important mechanism for the disposal of misfolded and damaged proteins in mammalian cells. Under mechanical stress, the cochaperone cooperates with the small heat shock protein HSPB8 and the cytoskeleton-associated protein SYNPO2 to degrade force-unfolded forms of the actin-crosslinking protein filamin. This is essential for muscle maintenance in flies, fish, mice and men. Here, we identify the serine/threonine protein kinase 38 (STK38), which is part of the Hippo signaling network, as a novel interactor of BAG3. STK38 was previously shown to facilitate cytoskeleton assembly and to promote mitophagy as well as starvation and detachment induced autophagy. Significantly, our study reveals that STK38 exerts an inhibitory activity on BAG3-mediated autophagy. Inhibition relies on a disruption of the functional interplay of BAG3 with HSPB8 and SYNPO2 upon binding of STK38 to the cochaperone. Of note, STK38 attenuates CASA independently of its kinase activity, whereas previously established regulatory functions of STK38 involve target phosphorylation. The ability to exert different modes of regulation on central protein homeostasis (proteostasis) machineries apparently allows STK38 to coordinate the execution of diverse macroautophagy pathways and to balance cytoskeleton assembly and degradation.
Background/Aims: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli.
Methods: hSK2-tg mice were generated by using a Quick KnockinR strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by 3H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA.
Results: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1.
Conclusion: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.
Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis.
Biological ageing is a degenerative and irreversible process, ultimately leading to death of the organism. The process is complex and under the control of genetic, environmental and stochastic traits. Although many theories have been established during the last decades, none of these are able to fully describe the complex mechanisms, which lead to ageing. Generally, biological processes and environmental factors lead to molecular damage and an accumulation of impaired cellular components. In contrast, counteracting surveillance systems are effective, including repair, remodelling and degradation of damaged or impaired components, respectively. Nevertheless, at some point these systems are no longer effective, either because the increasing amount of molecular damages can not longer be removed efficiently or because the repairing and removing mechanisms themselves become affected by impairing effects. The organism finally declines and dies. To investigate and to understand these counteracting mechanisms and the complex interplay of decline and maintenance, holistic and systems biological investigations are required. Hence, the processes which lead to ageing in the fungal model organism Podospora anserina, had been analysed using different advanced bioinformatics methods. In contrast to many other ageing models, P. anserina exhibits a short lifespan, a less biochemical complexity and it provides a good accessibility for genetic manipulations.
To achieve a general overview on the different biochemical processes, which are affected during ageing in P. anserina, an initial comprehensive investigation was applied, which aimed to reveal genes significantly regulated and expressed in an age-dependent manner. This investigation was based on an age-dependent transcriptome analysis. Sophisticated and comprehensive analyses revealed different age-related pathways and indicated that especially autophagy may play a crucial role during ageing. For example, it was found that the expression of autophagy-associated genes increases in the course of ageing.
Subsequently, to investigate and to characterise the autophagy pathway, its associated single components and their interactions, Path2PPI, a new bioinformatics approach, was developed. Path2PPI enables the prediction of protein-protein interaction networks of particular pathways by means of a homology comparison approach and was applied to construct the protein-protein interaction network of autophagy in P. anserina.
The predicted network was extended by experimental data, comprising the transcriptome data as well as newly generated protein-protein interaction data achieved from a yeast two-hybrid analysis. Using different mathematical and statistical methods the topological properties of the constructed network had been compared with those of randomly generated networks to approve its biological significance. In addition, based on this topological and functional analysis, the most important proteins were determined and functional modules were identified, which correspond to the different sub-pathways of autophagy. Due to the integrated transcriptome data the autophagy network could be linked to the ageing process. For example, different proteins had been identified, which genes are continuously up- or down-regulated during ageing and it was shown for the first time that autophagy-associated genes are significantly often co-expressed during ageing.
The presented biological network provides a systems biological view on autophagy and enables further studies, which aim to analyse the relationship of autophagy and ageing. Furthermore, it allows the investigation of potential methods for intervention into the ageing process and to extend the healthy lifespan of P. anserina as well as of other eukaryotic organisms, in particular humans.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.
Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.
Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.
Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.
The family of lysosome-associated membrane proteins (LAMP) includes the ubiquitously expressed LAMP1 and LAMP2, which account for half of the proteins in the lysosomal membrane. Another member of the LAMP family is LAMP3, which is expressed only in certain cell types and differentiation stages. LAMP3 expression is linked with poor prognosis of certain cancers, and the locus where it is encoded was identified as a risk factor for Parkinson's disease (PD). Here, we investigated the role of LAMP3 in the two main cellular degradation pathways, the proteasome and autophagy. LAMP3 mRNA was not detected in mouse models of PD or in the brain of human patients. However, it was strongly induced upon proteasomal inhibition in the neuroblastoma cell line SH-SY5Y. Induction of LAMP3 mRNA following proteasomal inhibition was dependent on UPR transcription factor ATF4 signaling and induced autophagic flux. Prevention of LAMP3 induction enhanced apoptotic cell death. In summary, these data demonstrate that LAMP3 regulation as part of the UPR contributes to protein degradation and cell survival during proteasomal dysfunction. This link between autophagy and the proteasome may be of special importance for the treatment of tumor cells with proteasomal inhibitors.
Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.