Carlo Rodolfo

Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH

In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (Refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5´→3´ helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

‘Tissue’ transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK‐N‐BE (2)) in which the tTG‐expression was driven by a constitutive promoter. Furthermore, a tet‐off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared ‘sensitized’ to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial‐mediated apoptosis by stauro‐ sporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a ‘sensitizer’ towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Inhibition of “Tissue” Transglutaminase Increases Cell Survival by Preventing Apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of “tissue” transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca2+-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.

The adenine nucleotide translocator 1 acts as a type 2 transglutaminase substrate: implications for mitochondrial-dependent apoptosis.

In this study we provide in vitro and in vivo evidence showing that the protein disulphide isomerase (PDI) activity of type 2 transglutaminase (TG2) regulates the correct assembly and function of the mitochondrial ADP/ATP transporter adenine nucleotide translocator 1 (ANT1). We demonstrate, by means of biochemical and morphological analyses, that ANT1 and TG2 physically interact in the mitochondria. Under physiological conditions, TG2s PDI activity regulates the ADP/ATP transporter function by controlling the oligomerization of ANT1. In fact, mitochondria isolated from hearts of TG2−/− mice exhibit increased polymerization of ANT1, paralleled by an enhanced ADP/ATP carrier activity, as compared to mitochondria belonging to TG2+/+ mice. Interestingly, upon cell-death induction, ANT1 becomes a substrate for TG2s cross-linking activity and the lack of TG2 results in a reduction of apoptosis as well as in a marked sensitivity to the ADP/ATP exchange inhibition by atractyloside. These findings suggest a complex TG2-dependent regulation of the ADP/ATP transporter and reveal new important avenues for its potential applications in the treatment of some mitochondrial-dependent diseases, including cardiovascular and neurodegenerative diseases.

Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation

ULK1 is a key kinase in autophagy initiation. Nazio et al. demonstrate that the E3 ubiquitin ligase NEDD4L targets ULK1 for degradation soon after autophagy induction, whereas a simultaneous ULK1 mRNA transcription is needed for priming subsequent rounds of autophagy.

Autophagy in stem and progenitor cells

Autophagy is a highly conserved cellular process, responsible for the degradation and recycling of damaged and/or outlived proteins and organelles. This is the major cellular pathway, acting throughout the formation of cytosolic vesicles, called autophagosomes, for the delivering to lysosome. Recycling of cellular components through autophagy is a crucial step for cell homeostasis as well as for tissue remodelling during development. Impairment of this process has been related to the pathogenesis of various diseases, such as cancer and neurodegeneration, to the response to bacterial and viral infections, and to ageing. The ability of stem cells to self-renew and differentiate into the mature cells of the body renders this unique type of cell highly crucial to development and tissue renewal, not least in various diseases. During the last two decades, extensive knowledge about autophagy roles and regulation in somatic cells has been acquired; however, the picture about the role and the regulation of autophagy in the different types of stem cells is still largely unknown. Autophagy is a major player in the quality control and maintenance of cellular homeostasis, both crucial factors for stem cells during an organism’s life. In this review, we have highlighted the most significant advances in the comprehension of autophagy regulation in embryonic and tissue stem cells, as well as in cancer stem cells and induced pluripotent cells.

Proteomic analysis of mitochondrial dysfunction in neurodegenerative diseases

Alzheimer’s, Parkinson’s and Huntington’s disease, and amyotrophic lateral sclerosis are the most relevant neurodegenerative syndromes worldwide. The identification of the etiology and additional factors contributing to the onset and progression of these diseases is of great importance in order to develop both preventive and therapeutic intervention. A common feature of these pathologies is the formation of aggregates, containing mutated and/or misfolded proteins, in specific subsets of neurons, which progressively undergo functional impairment and die. The relationship between protein aggregation and the molecular events leading to neurodegeneration has not yet been clarified. In the last decade, several lines of evidence pointed to a major role for mitochondrial dysfunction in the onset of these pathologies. Here, we review how proteomics has been applied to neurodegenerative diseases in order to characterize the relationship existing between protein aggregation and mitochondrial alterations. Moreover, we highlight recent advances in the use of proteomics to identify protein modifications caused by oxidative stress. Future developments in this field are expected to significantly contribute to the full comprehension of the molecular mechanisms at the heart of neurodegeneration.

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293TG) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.

In vivo evaluation of type 2 transglutaminase contribution to the metastasis formation in melanoma

One of the most relevant problems in tumour treatment resides on the ability of the tumour to form metastasis and disseminate among the organism. The formation of metastases is a complex process, which requires the action of various effectors, not yet completely identified. The analysis of various types of tumours revealed a complex picture about the relationship between type 2 transglutaminase (TG2) expression and outcome and/or metastatic potential of the tumour itself. In some tumours, the transition to a highly invasive state is paralleled by an up-regulation of TG2 expression and/or activity while in some other a down-regulation has been reported. In addition, host tissues seem to react to tumour invasion by up-regulating TG2 expression. In order to analyse whether TG2 might be involved in the metastatic process in melanoma, we studied the metastases formation and development by means of the B16-F10 murine melanoma cell line and with TG2−/− mice as experimental model. Our results indicate that TG2 absence in the host is a favouring condition for the formation and development of the metastasis, while the presence of TG2 in the tumour’s cell might be requested for the development of the metastasis.

Tissue transglutaminase expression in HIV-infected cells: an enzyme with an antiviral effect?

Abstract: The cytopathic effect of HIV has been shown to be associated with the induction of apoptosis and the inhibition of proliferation of T cells. However, the cellular and molecular mechanisms at the basis of the dramatic immune cell loss caused by HIV in patients suffering from acquired immunodeficient syndrome (AIDS), are not yet fully established. We demonstrated that “tissue” transglutaminase (tTG) gene expression is induced in the immune system of seropositive individuals (peripheral blood mononuclear cells and lymph nodes). tTG is a multifunctional protein involved in a variety of fundamentally important cellular functions, in addition to cell death by apoptosis. The presence of high tTG levels in immune‐competent cells of HIV+ persons might exert an important role in HIV‐infection by influencing viral production. We propose that, in addition to its multiple functions, tTG might interfere with HIV replication by altering the viral mRNA trafficking between the nucleus and the cytoplasm. This effect might be due to its specific interaction with eIF5A, a cellular partner of HIV Rev protein, which is essential for HIV replication in immune‐competent cells. Given the presence of high tTG levels in HIV+ individuals, it would be of interest to pursue the potential role of this multifunctional protein in the development of strategies aimed at the pharmacologic regulation of HIV production.