The Role of Rab Proteins in Mitophagy: Insights into Neurodegenerative Diseases.

Mitochondrial dysfunction and vesicular trafficking alterations have been implicated in the pathogenesis of several neurodegenerative diseases. It has become clear that pathogenetic pathways leading to neurodegeneration are often interconnected. Indeed, growing evidence suggests a concerted contribution of impaired mitophagy and vesicles formation in the dysregulation of neuronal homeostasis, contributing to neuronal cell death. Among the molecular factors involved in the trafficking of vesicles, Ras analog in brain (Rab) proteins seem to play a central role in mitochondrial quality checking and disposal through both canonical PINK1/Parkin-mediated mitophagy and novel alternative pathways. In turn, the lack of proper elimination of dysfunctional mitochondria has emerged as a possible causative/early event in some neurodegenerative diseases. Here, we provide an overview of major findings in recent years highlighting the role of Rab proteins in dysfunctional mitochondrial dynamics and mitophagy, which are characteristic of neurodegenerative diseases. A further effort should be made in the coming years to clarify the sequential order of events and the molecular factors involved in the different processes. A clear cause-effect view of the pathogenetic pathways may help in understanding the molecular basis of neurodegeneration.

Demonstration of fibrinogen-FcRn binding at acidic pH by means of Fluorescence Correlation Spectroscopy.

The neonatal Fc receptor (FcRn) interacts with IgG and albumin at acidic pH within endosomes, thus protecting these plasma proteins from degradation. Recently, we proposed fibrinogen as a new binding partner of FcRn. This work was aimed at providing a direct demonstration of FcRn-fibrinogen binding at acidic pH by Fluorescence Correlation Spectroscopy. The increase in diffusion time between free and fibrinogen-bound FITC-labelled FcRn was assumed as the binding indicator. We observed that, at acidic pH (pH = 5.3), FcRn diffusion time shifted from ≈730 µs (FITC-labelled FcRn alone) to >1200 µs (FITC-labelled FcRn added with fibrinogen). A similar trend was exhibited by albumin, a known FcRn interactor, while no significant variations in diffusion time were observed upon incubation with catalase as negative control. Our results demonstrate a binding interaction between fibrinogen, one of the most abundant plasma proteins, and FcRn, a receptor involved in the regulation of the levels of IgG and albumin. This interaction is likely responsible for fibrinogen protection from intracellular degradation and recycling in plasma. Fibrinogen is crucial not only in haemostasis but also in acute inflammatory response and in some pathological conditions. The interaction with FcRn can influence not only the levels of fibrinogen in plasma and other tissues, but also the levels of other FcRn binding partners, among which are some plasma proteins of clinical relevance.

C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of the corticospinal motor neurons, which ultimately leads to death. The repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) represents the most common genetic cause of ALS and it is also involved in the pathogenesis of other neurodegenerative disorders. To offer insights into C9ORF72-mediated pathogenesis, we quantitatively analyzed the proteome of patient-derived primary skin fibroblasts from ALS patients carrying the C9ORF72 mutation compared with ALS patients who tested negative for it. Differentially expressed proteins were identified, used to generate a protein-protein interaction network and subjected to a functional enrichment analysis to unveil altered molecular pathways. ALS patients were also compared with patients affected by frontotemporal dementia carrying the C9ORF72 repeat expansion. As a result, we demonstrated that the molecular pathways mainly altered in fibroblasts (e.g., protein homeostasis) mirror the alterations observed in C9ORF72-mutated neurons. Moreover, we highlighted novel molecular pathways (nuclear and mitochondrial transports, vesicle trafficking, mitochondrial bioenergetics, glucose metabolism, ER-phagosome crosstalk and Slit/Robo signaling pathway) which might be further investigated as C9ORF72-specific pathogenetic mechanisms. Data are available via ProteomeXchange with the identifier PXD023866.

Proteostasis and Proteotoxicity in the Network Medicine Era.

Neurodegenerative proteinopathies are complex diseases that share some pathogenetic processes. One of these is the failure of the proteostasis network (PN), which includes all components involved in the synthesis, folding, and degradation of proteins, thus leading to the aberrant accumulation of toxic protein aggregates in neurons. The single components that belong to the three main modules of the PN are highly interconnected and can be considered as part of a single giant network. Several pharmacological strategies have been proposed to ameliorate neurodegeneration by targeting PN components. Nevertheless, effective disease-modifying therapies are still lacking. In this review article, after a general description of the PN and its failure in proteinopathies, we will focus on the available pharmacological tools to target proteostasis. In this context, we will discuss the main advantages of systems-based pharmacology in contrast to the classical targeted approach, by focusing on network pharmacology as a strategy to innovate rational drug design.

Mitochondrial Proteins in the Development of Parkinson's Disease.

Parkinson's disease (PD) is a multifactorial disorder whose etiology is not completely understood. Strong evidences suggest that mitochondrial impairment and altered mitochondrial disposal play a key role in the development of this pathology. Here we show this association in both genetic and sporadic forms of the disease. Moreover, we describe the mitochondrial dysfunctions in toxin-induced models of PD, thus highlighting the importance of environmental factors in the onset of this pathology. In particular, we focus our attention on mitochondrial dynamics, mitochondrial biogenesis, and mitophagy and explain how their impairment could have a negative impact on dopaminergic neurons function and survival. Lastly, we aim at clarifying the important role played by proteomics in this field of research, proteomics being a global and unbiased approach suitable to unravel alterations of the molecular pathways in multifactorial diseases.

Update of the Functional Mitochondrial Human Proteome Network.

Because of the pivotal role of mitochondrial alterations in several diseases, the Human Proteome Organization (HUPO) has promoted in recent years an initiative to characterize the mitochondrial human proteome, the mitochondrial human proteome project (mt-HPP). Here we generated an updated version of the functional mitochondrial human proteome network, made by nodes (mitochondrial proteins) and edges (gold binary interactions), using data retrieved from neXtProt, the reference database for HPP metrics. The principal new concept suggested was the consideration of mitochondria-associated proteins (first interactors), which may influence mitochondrial functions. All of the proteins described as mitochondrial in the sublocation or the GO Cellular Component sections of neXtProt were considered. Their other subcellular and submitochondrial localizations have been analyzed. The network represents the effort to collect all of the high-quality binary interactions described so far for mitochondrial proteins and the possibility for the community to reuse the information collected. As a proof of principle, we mapped proteins with no function, to speculate on their role by the background knowledge of their interactors, and proteins described to be involved in Parkinson's Disease, a neurodegenerative disorder, where it is known that mitochondria play a central role.

Neonatal Fc receptor is involved in the protection of fibrinogen after its intake in peripheral blood mononuclear cells.

BACKGROUND: Fibrinogen is a central player in the blood coagulation cascade and one of the most abundant plasma proteins. This glycoprotein also triggers important events (e.g., cell spreading, the respiratory burst and degranulation) in neutrophil cells via a αMß2 integrin-mediated binding to the cell surface. Yet, little is known about the interaction of fibrinogen with leukocytes other than neutrophils or stimulated monocytes, although high amounts of fibrinogen protein can also be found in lymphocytes, particularly in T-cells. The aim of the present work is to unveil the dynamics and the function of fibrinogen intake in T-cells. METHODS: Using the Jurkat cell line as a T-cells model we performed fibrinogen intake/competition experiments. Moreover, by means of a targeted gene knock-down by RNA-interference, we investigated the dynamics of the intake mechanism. RESULTS: Here we show that (i) fibrinogen, although not expressed in human peripheral blood mononuclear cells, can be internalized by these cells; (ii) fibrinogen internalization curves show a hyperbolic behavior, which is affected by the presence of serum in the medium, (iii) FITC-conjugated fibrinogen is released and re-internalized by adjacent cells, (iv) the presence of human serum albumin (HSA) or immunoglobulin G (IgG), which are both protected from intracellular degradation by the interaction with the neonatal Fc receptor (FcRn), results in a decreased amount of internalized fibrinogen, and (v) FcRn-knockdown affects the dynamics of fibrinogen internalization. CONCLUSIONS: We demonstrated here for the first time that fibrinogen can be internalized and released by T-lymphocyte cells. Moreover, we showed that the presence of serum, HSA or IgG in the culture medium results in a reduction of the amount of internalized fibrinogen in these cells. Thus, we obtained experimental evidence for the expression of FcRn in T-lymphocyte cells and we propose this receptor as involved in the protection of fibrinogen from intracellular lysosomal degradation.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Chronic Δ8-THC Exposure Differently Affects Histone Modifications in the Adolescent and Adult Rat Brain.

Adolescence represents a vulnerable period for the psychiatric consequences of delta9-tetrahydrocannabinol (Δ8-THC) exposure, however, the molecular underpinnings of this vulnerability remain to be established. Histone modifications are emerging as important epigenetic mechanisms involved in the etiopathogenesis of psychiatric diseases, thus, we investigated the impact of chronic Δ8-THC exposure on histone modifications in different brain areas of female rats. We checked histone modifications associated to both transcriptional repression (H3K9 di- and tri-methylation, H3K27 tri-methylation) and activation (H3K9 and H3K14 acetylation) after adolescent and adult chronic Δ8-THC exposure in the hippocampus, nucleus accumbens, and amygdala. Chronic exposure to increasing doses of Δ8-THC for 11 days affected histone modifications in a region- and age-specific manner. The primary effect in the adolescent brain was represented by changes leading to transcriptional repression, whereas the one observed after adult treatment led to transcriptional activation. Moreover, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the Δ8-THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to Δ8-THC adverse effects.

Dopamine induces mitochondrial depolarization without activating PINK1-mediated mitophagy.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. PD mostly occurs sporadically and its cause remains unknown, nevertheless the discovery of familiar forms of PD, characterized by mutations of genes encoding proteins associated with mitochondria homeostasis, suggests a strong implication of the mitochondrial quality control system in PD. We investigated the effect of dopamine cytosolic accumulation in undifferentiated SH-SY5Y cells, an in vitro model widely used to reproduce impairment of dopamine homeostasis, an early step in PD pathogenesis. A strong depolarization of the mitochondrial membrane was observed after dopamine exposure. Nevertheless, mitochondrial network resulted to assume a peculiar morphology with a distinct pattern of OPA1 and MFN1, key regulators of mitochondrial dynamics. Moreover, selective elimination of dysfunctional mitochondria did not take place, suggesting an impairment of the mitophagic machinery induced by dopamine. Indeed, PINK1 did not accumulate on the outer mitochondrial membrane, nor was parkin recruited to depolarized mitochondria. Altogether, our results indicate that an improper handling of dysfunctional mitochondria may be a leading event in PD pathogenesis. Impaired dopamine (DA) homeostasis and oxidative stress play a key role in the pathogenesis of Parkinson's disease. Free cytosolic dopamine undergoes spontaneous oxidation and generates semiquinonic and quinonic species (DAQ) with the concurrent production of reactive oxygen species (ROS). Dopamine dissipates mitochondrial potential (Δψm ) with a peculiar alteration of the mitochondrial network. However, PINK1-dependent mitophagy is not activated by dopamine toxicity and dysfunctional mitochondria accumulate inside the cell.

A systems biology-led insight into the role of the proteome in neurodegenerative diseases.

INTRODUCTION: Multifactorial disorders are the result of nonlinear interactions of several factors; therefore, a reductionist approach does not appear to be appropriate. Proteomics is a global approach that can be efficiently used to investigate pathogenetic mechanisms of neurodegenerative diseases. AREAS COVERED: Here, we report a general introduction about the systems biology approach and mechanistic insights recently obtained by over-representation analysis of proteomics data of cellular and animal models of Alzheimer's disease, Parkinson's disease and other neurodegenerative disorders, as well as of affected human tissues. Expert commentary: As an inductive method, proteomics is based on unbiased observations that further require validation of generated hypotheses. Pathway databases and over-representation analysis tools allow researchers to assign an expectation value to pathogenetic mechanisms linked to neurodegenerative diseases. The systems biology approach based on omics data may be the key to unravel the complex mechanisms underlying neurodegeneration.

Altered dopamine homeostasis differentially affects mitochondrial voltage-dependent anion channels turnover.

Altered dopamine homeostasis plays a key role in the pathogenesis of Parkinson's disease. The generation of reactive oxygen species by spontaneous dopamine oxidation impairs mitochondrial function, causing in turn an enhancement of oxidative stress. Recent findings have highlighted the role of mitochondrial outer membrane proteins in the regulation of the correct disposal of damaged mitochondria. Here, we report the effect of altered dopamine homeostasis on the mitochondrial functionality in human neuroblastoma SH-SY5Y cells, a cellular model widely used to reproduce impaired dopamine homeostasis. We observed that dopamine significantly and relevantly reduces VDAC1 and VDAC2 levels without any change in the mRNA levels. Although mitochondria are depolarized by dopamine and mitochondrial calcium influx is reduced, dysfunctional mitochondria are not removed by mitophagy as it would be expected. Thus, alteration of dopamine homeostasis induces a mitochondrial depolarization not counteracted by the mitophagy quality control. As a consequence, the elimination of VDACs may contribute to the altered mitochondrial disposal in PD pathogenesis, thus enhancing the role of oxidative stress.

Verification of a Parkinson's disease protein signature in T-lymphocytes by multiple reaction monitoring.

Diagnosis of Parkinson's disease, the second most common neurodegenerative disease, is based on the appearance of motor symptoms. A panel of protein biomarkers in the T-lymphocyte proteome was previously proposed as a Parkinson's disease signature. Here, we designed an LC-MS based method to quantitatively evaluate this protein signature by multiple reaction monitoring (MRM) in T-lymphocytes and peripheral blood mononuclear cells from a new cohort of nine patients with Parkinson's disease and nine unaffected subjects. Patients were classified using the discriminant function obtained from two-dimensional electrophoresis and protein amounts measured by MRM, thus assigning seven controls out of nine as true negatives and nine patients out of nine as true positives. A good discriminant power was obtained by selecting a subset of peptides from the protein signature, with an area under the receiver operating characteristic curve of 0.877. A similar result is achieved by evaluating all peptides of a selected panel of proteins (gelsolin, moesin, septin-6, twinfilin-2, lymphocyte-specific protein 1, vimentin, transaldolase), with an area under the curve of 0.840. Conversely, the signature was not able to classify the enrolled subjects when evaluated in whole mononuclear cells. Overall, this report shows the portability of the proposed method to a large-scale clinical validation study.

Multiple isoforms and differential allelic expression of CHRNA5 in lung tissue and lung adenocarcinoma.

CHRNA5 gene expression variation may play a role in individual susceptibility to lung cancer. Analysis of CHRNA5 transcripts expressed in normal lung tissue detected the full-length transcript (isoform-1) and four splicing transcripts (isoform-2 to isoform-5), derived from the recognition of other splice sites in exon 5. Isoforms-2, -3 and -4 were found by protein modeling to form a completely folded, potentially functional extracellular domain and were observed at the protein level, whereas isoform-5 lacked a consistent part of the distorted ß sandwich and was not seen at the protein level. Only isoform-1 appeared to encode a complete, functional subunit able to fulfill the ion channel function. We previously reported that CHRNA5 expression is associated with genetic polymorphisms at this locus and that three haplotypes in its promoter region show functional regulation in vitro. Analysis of differential allelic expression (DAE) of three single nucleotide polymorphisms (rs503464, rs55853698 and rs55781567) tagging the expression haplotypes of the CHRNA5 promoter indicated statistically significant DAE at rs55853698 and rs55781567, in both normal lung and lung adenocarcinoma. Overall, our findings provide evidence for the presence of multiple CHRNA5 messenger RNA (mRNA) isoforms that may modulate the multimeric nicotine receptor and cis-regulatory variations in the CHRNA5 locus that act in vivo in the control of CHRNA5 mRNA expression, in normal lung tissue and in lung adenocarcinoma.

Molecular phylogenetic analyses of albuminoids reveal the molecular evolution of allosteric properties.

Serum albumin, α-fetoprotein, afamin (also named α-albumin and vitamin E binding protein), and vitamin D binding protein are members of the albuminoid superfamily. Albuminoids are plasma proteins characterized by a marked ability for ligand binding and transport. Here, a focused phylogenetic analysis of sequence evolution by maximum likelihood of fatty acid binding sites FA1-FA7 of mammalian albuminoids reveals that the FA1, FA2, and FA3+FA4 sites in serum albumins have evolved from the most recent common ancestor through an intermediate that has originated the α-fetoprotein and afamin clades. The same topology has been observed for the whole protein sequences, for the sequences of all the fatty acid binding sites (FA1-FA7) taken together, and for the allosteric core corresponding to residues 1-303 of human serum albumin. The quantitative divergence analysis indicates that the ligand binding cleft corresponding to the FA2 site could be the main determinant of allosteric properties of serum albumins only. In fact, this binding cleft is structurally not effective in vitamin D binding proteins, whereas key residues that serve to allocate the allosteric effectors are not present in afamins and α-fetoproteins.

Neurodegenerative disorders: From clinicopathology convergence to systems biology divergence.

Neurodegenerative diseases are multifactorial. This means that several genetic, epigenetic, and environmental factors contribute to their emergence. Therefore, for the future management of these highly prevalent diseases, it is necessary to change perspective. If a holistic viewpoint is assumed, the phenotype (the clinicopathological convergence) emerges from the perturbation of a complex system of functional interactions among proteins (systems biology divergence). The systems biology top-down approach starts with the unbiased collection of sets of data generated through one or more -omics techniques and has the aim to identify the networks and the components that participate in the generation of a phenotype (disease), often without any available a priori knowledge. The principle behind the top-down method is that the molecular components that respond similarly to experimental perturbations are somehow functionally related. This allows the study of complex and relatively poorly characterized diseases without requiring extensive knowledge of the processes under investigation. In this chapter, the use of a global approach will be applied to the comprehension of neurodegeneration, with a particular focus on the two most prevalent ones, Alzheimer's and Parkinson's diseases. The final purpose is to distinguish disease subtypes (even with similar clinical manifestations) to launch a future of precision medicine for patients with these disorders.

Shared and Unique Disease Pathways in Amyotrophic Lateral Sclerosis and Parkinson's Disease Unveiled in Peripheral Blood Mononuclear Cells.

Recent evidence supports an association between amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Indeed, prospective population-based studies demonstrated that about one-third of ALS patients develop parkinsonian (PK) signs, even though different neuronal circuitries are involved. In this context, proteomics represents a valuable tool to identify unique and shared pathological pathways. Here, we used two-dimensional electrophoresis to obtain the proteomic profile of peripheral blood mononuclear cells (PBMCs) from PD and ALS patients including a small cohort of ALS patients with parkinsonian signs (ALS-PK). After the removal of protein spots correlating with confounding factors, we applied a sparse partial least square discriminant analysis followed by recursive feature elimination to obtain two protein classifiers able to discriminate (i) PD and ALS patients (30 spots) and (ii) ALS-PK patients among all ALS subjects (20 spots). Functionally, the glycolysis pathway was significantly overrepresented in the first signature, while extracellular interactions and intracellular signaling were enriched in the second signature. These results represent molecular evidence at the periphery for the classification of ALS-PK as ALS patients that manifest parkinsonian signs, rather than comorbid patients suffering from both ALS and PD. Moreover, we confirmed that low levels of fibrinogen in PBMCs is a characteristic feature of PD, also when compared with another movement disorder. Collectively, we provide evidence that peripheral protein signatures are a tool to differentially investigate neurodegenerative diseases and highlight altered biochemical pathways.

Dopaminergic therapies modulate the T-CELL proteome of patients with Parkinson's disease.

Dopamine receptor agonists and L-dihydroxyphenylalanine (L-DOPA) counteract dopamine loss in the striatum and are therefore used in the treatment of Parkinson's disease (PD). T-Lymphocytes express some features of the dopaminergic system, and their function or activation might be regulated by dopaminergic treatments. Two-dimensional electrophoresis of total protein extract from T-lymphocytes was performed to identify therapy-induced proteome changes in T-cells of 17 patients with PD. Specific protein level alterations were further validated by Western blotting. Of 17 enrolled patients, 11 were treated with different doses of L-DOPA; in this group, we found that the levels of two spots, corresponding to ATP synthase subunit ß and proteasome subunit ß type-2, correlated linearly with the L-DOPA daily dose. Moreover, we identified seven proteins (prolidase, actin-related protein 2, F-actin-capping protein subunit ß, tropomyosin α-3 chain, proteasome activator complex subunit 1, peroxiredoxin 6, and a glyceraldehyde-3-phosphate dehydrogenase isoform) whose levels were significantly different in patients treated with dopamine agonists. These findings demonstrate that dopaminergic stimulation has important effects on T-cell proteome in patients under long-term treatment. Therefore, therapies acting on the dopaminergic system may have additional effects on the immune system.

Current Insights on Neurodegeneration by the Italian Proteomics Community.

The growing number of patients affected by neurodegenerative disorders represents a huge problem for healthcare systems, human society, and economics. In this context, omics strategies are crucial for the identification of molecular factors involved in disease pathobiology, and for the discovery of biomarkers that allow early diagnosis, patients' stratification, and treatment response prediction. The integration of different omics data is a required step towards the goal of personalized medicine. The Italian proteomics community is actively developing and applying proteomics approaches to the study of neurodegenerative disorders; moreover, it is leading the mitochondria-focused initiative of the Human Proteome Project, which is particularly important given the central role of mitochondrial impairment in neurodegeneration. Here, we describe how Italian research groups in proteomics have contributed to the knowledge of many neurodegenerative diseases, through the elucidation of the pathobiology of these disorders, and through the discovery of disease biomarkers. In particular, we focus on the central role of post-translational modifications analysis, the implementation of network-based approaches in functional proteomics, the integration of different omics in a systems biology view, and the development of novel platforms for biomarker discovery for the high-throughput quantification of thousands of proteins at a time.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.