Proteomics and bioinformatics analyses identify novel cellular roles outside mitochondrial function for human miro GTPases

The human Miro GTPases (hMiros) have recently emerged as important mediators of mitochondrial transport and may significantly contribute to the development of disorders such as Alzheimer’s and schizophrenia. The hMiros represent two highly atypical members of the Ras superfamily, and exhibit several unique features: the presence of a GTPase domain at both the N-terminus and C-terminus, the presence of two calcium-binding EF-hand domains and localisation to the mitochondrial outer membrane. Here, elucidation of Miro GTPase signalling pathway components was achieved through the use of molecular biology, cell culture techniques and proteomics. An investigation of this kind has not been performed previously; we hoped, through these techniques, to enable the profiling and identification of pathways regulated by the human Miro GTPases. The results indicate several novel putative interaction partners for hMiro1 and hMiro2, including numerous proteins previously implicated in neurodegenerative pathways and the development of schizophrenia. Furthermore, we show that the N-terminal GTPase domain appears to fine-tune hMiro signalling, with GTP-bound versions of this domain associated with a diverse range of interaction partners in comparison to corresponding GDP-bound versions. Recent evidences suggest that human Miros participate in host–pathogen interactions with Vibrio Cholerae type III secretion proteins. We have undertaken a bioinformatics investigation to identify novel pathogenic effectors that might interact with Miros

Understanding miro GTPases: implications in the treatment of neurodegenerative disorders

The Miro GTPases represent an unusual subgroup of the Ras superfamily and have recently emerged as important mediators of mitochondrial dynamics and for maintaining neuronal health. It is now well-established that these enzymes act as essential components of a Ca2+-sensitive motor complex, facilitating the transport of mitochondria along microtubules in several cell types, including dopaminergic neurons. The Miros appear to be critical for both anterograde and retrograde mitochondrial transport in axons and dendrites, both of which are considered essential for neuronal health. Furthermore, the Miros may be significantly involved in the development of several serious pathological processes, including the development of neurodegen- erative and psychiatric disorders. In this review, we discuss the molecular structure and known mitochondrial functions of the Miro GTPases in humans and other organisms, in the context of neurodegenerative disease. Finally, we consider the potential human Miros hold as novel therapeutic targets for the treatment of such disease

DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis

Epidermal growth factor receptor (EGFR) gene alteration and protein overexpression in Malaysian triple-negative breast cancer (TNBC) cohort

Background: Epidermal growth factor receptor (EGFR) is a member of the ErbB family of tyrosine kinase receptor proteins that plays important roles in tumour cell survival and proliferation. EGFR has been reported to be overexpressed in up to 78% of triple-negative breast cancer (TNBC) cases suggesting it as a potential therapeutic target. The clinical trials of anti-EGFR agents in breast cancer showed low response rates. However, a subgroup of patients demonstrated response to EGFR inhibitors highlighting the necessity to stratify patients, who might benefit from effective combination therapy that could include anti EGFR-agents. Population variability in EGFR expression warrants systematic evaluation in specific populations. Purpose: To study EGFR alterations and expressions in a multi ethnic Malaysian TNBC patient cohort to determine the possibility of using anti-EGFR combinatorial therapy for this population. Patients and methods: In this study, we evaluated 58 cases of Malaysian TNBC patient samples for EGFR gene copy number alteration and EGFR protein overexpression using fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) methods, respectively. Results: EGFR protein overexpression was observed in about 30% while 15.5% displayed high EGFR copy number including 5.17% gene amplification and over 10% high polysomy. There is a positive correlation between EGFR protein overexpression and gene copy number and over expression of EGFR is observed in ten out of the 48 low copy number cases (20.9%) without gene amplification. Conclusion: This study provides the first glimpse of EGFR alterations and expressions in a multi ethnic Malaysian TNBC patient cohort emphasising the need for the nationwide large scale EGFR expression evaluation in Malaysia

Novel 1-hydroxypyridin-2-one metal chelators prevent and res-cue ubiquitin proteasomal-related neuronal injury in an in vitro model of Parkinson’s disease

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal de-generation in Parkinson's disease (PD); however, a link between these features remains unconfirmed. By using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxi-dative stress and promoting protein aggregation. We demonstrate the neuroprotec-tive potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds’ iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also de-monstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe3+ and display a 3:1 HOPO:Fe3+ complex formation at physiological pH. Reducing iron reactivity could prevent the demise of nigral dopaminergic neurons. We provide evidence that the lactacystin model presents with several neuropatho-logical hallmarks of PD related to iron dyshomeostasis and that the novel chelating compounds C6 and C9 can protect against lactacystin-related neurotoxicity

Low BACH2 Expression Predicts Adverse Outcome in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a highly variable clinical outcome. There are well‐established CLL prognostic biomarkers that have transformed treatment and improved the understanding of CLL biology. Here, we have studied the clinical significance of two crucial B cell regulators, BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) and BCL6 (B‐cell CLL/lymphoma 6), in a cohort of 102 CLL patients and determined the protein interaction networks that they participate in using MEC‐1 CLL cells. We observed that CLL patients expressing low levels of BCL6 and BACH2 RNA had significantly shorter overall survival (OS) than high BCL6‐ and BACH2‐expressing cases. Notably, their low expression specifically decreased the OS of immunoglobulin heavy chain variable region‐mutated (IGHV‐M) CLL patients, as well as those with 11q and 13q deletions. Similar to the RNA data, a low BACH2 protein expression was associated with a significantly shorter OS than a high expression. There was no direct interaction observed between BACH2 and BCL6 in MEC‐1 CLL cells, but they shared protein networks that included fifty different proteins. Interestingly, a prognostic index (PI) model that we generated, using integrative risk score values of BACH2 RNA expression, age, and 17p deletion status, predicted patient outcomes in our cohort. Taken together, these data have shown for the first time a possible prognostic role for BACH2 in CLL and have revealed protein interaction networks shared by BCL6 and BACH2, indicating a significant role for BACH2 and BCL6 in key cellular processes, including ubiquitination mediated B‐cell receptor functions, nucleic acid metabolism, protein degradation, and homeostasis in CLL biology

Regulator of G-Protein Signaling 14 (RGS14) Is a Selective H-Ras Effector

Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Ga-mediated GTP hydrolysis (‘‘GTPase-accelerating proteins’’ or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Ga GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.Methodology/Principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co- transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor- mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.Conclusions/Significance: In cells, RGS14 facilitates the formation of a selective Ras?GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras- binding domain architecture with RGS14

Atypical GTPases as drug targets.

The Ras GTPases are the founding members of large Ras superfamily, which constitutes more than 150 of these important class of enzymes. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. There are a number of GTPases that have been identified recently, which do not confine to this prototype termed as "atypical GTPases" but have proved to play a remarkable role in vital cellular functions. In this review, we provide an overview of the crucial physiological functions mediated by RGK and Centaurin class of multi domain atypical GTPases. Moreover, the recently available atypical GTPase structures of the two families, regulation, physiological functions and their critical roles in various diseases will be discussed. In summary, this review will highlight the emerging atypical GTPase family which allows us to understand novel regulatory mechanisms and thus providing new avenues for drug discovery programs

Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism

Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are “atypical” Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be “relic” domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an “internal arginine finger”, allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms