Volume 396, Issue 8

Ras is a prototypical small G-protein and a central regulator of growth, proliferation and differentiation processes in virtually every nucleated cell. As such, Ras becomes engaged and activated by multiple growth factors, mitogens, cytokines or adhesion receptors. Ras activation comes about by changes in the steady-state equilibrium between the inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states of Ras, resulting in the mostly transient accumulation of Ras-GTP. Three decades of intense Ras research have disclosed various families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as the two principal regulatory elements of the Ras-GDP/GTP loading status. However, with the possible exception of the GEF Sos, we still have only a rudimentary knowledge of the precise role played by many GEF and GAP members in the signalling network upstream of Ras. As for GAPs, we even lack the fundamental understanding of whether they function as genuine signal transducers in the context of growth factor-elicited Ras activation or rather act as passive modulators of the Ras-GDP/GTP cycle. Here we sift through the large body of Ras literature and review the relevant data for understanding the participation and precise role played by GEFs and GAPs in the process of Ras activation.

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

Cathepsin S is a member of the cysteine cathepsin protease family. It is a lysosomal protease which can promote degradation of damaged or unwanted proteins in the endo-lysosomal pathway. Additionally, it has more specific roles such as MHC class II antigen presentation, where it is important in the degradation of the invariant chain. Unsurprisingly, mis-regulation has implicated cathepsin S in a variety of pathological processes including arthritis, cancer, and cardiovascular disease, where it becomes secreted and can act on extracellular substrates. In comparison to many other cysteine cathepsin family members, cathepsin S has uniquely restricted tissue expression and is more stable at a neutral pH, which supports its involvement and importance in localised disease microenvironments. In this review, we examine the known involvement of cathepsin S in disease, particularly with respect to recent work indicating its role in mediating pain, diabetes, and cystic fibrosis. We provide an overview of current literature with regards cathepsin S as a therapeutic target, as well as its role and potential as a predictive diagnostic and/or prognostic marker in these diseases.

Sox2 is well known for its functions in embryonic stem (ES) cell pluripotency, maintenance, and self-renewal, and it is an essential factor in generating inducible pluripotent stem (iPS) cells. It also plays an important role in development and adult tissue homeostasis of different tissues, especially the central nervous system. Increasing evidence has shown that aging is a stemness-related process in which Sox2 is also implicated as a key player, especially in the neural system. These distinct roles that Sox2 plays involve delicate regulatory networks consisting of other master transcription factors, microRNAs and signaling pathways. Additionally, the expression level of Sox2 can also be modulated transcriptionally, translationally or post-translationally. Here we will mainly review the roles of Sox2 in stem cell related development, homeostasis maintenance, aging processes, and the underlying molecular mechanisms involved.

Human discs large (DLG1) has been demonstrated to be involved in cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 expression are not fully understood. This is relevant as DLG1 is lost during the later stages of malignant progression. We initiated a series of studies to analyse the mechanisms regulating DLG1 expression. We have previously reported the identification of an alternative splicing event in the 5′ untranslated region (5′-UTR) of DLG1 mRNA that generates transcripts with two different 5′-UTR (short and large 5′-UTR variants). In this study, we further examined the impact of the DLG1 transcription and the role of the differential expression of the alternative 5′-UTRs on DLG1 protein levels. We analysed these mechanisms during cell processes like differentiation, cell cycle progression and cell-cell contact formation, where the importance of DLG1 activities was previously established. The data presented in this report suggest that the transcriptional regulation of DLG1 strongly contributes to DLG1 abundance and that differential expression of alternative 5′-UTRs with different translational properties, also cooperates, depending on the cell type and cell situation. This study provides new evidence for understanding the transcriptional regulation of DLG1 and the changes in DLG1 expression during different biological processes.

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni 2+ -affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

Despite the effectiveness of the anti-EGFR chimeric antibody (mAb), cetuximab, in treating nasopharyngeal carcinoma (NPC), its efficacy remains variable and often modest. In this study, a full human dual targeted anti-EGFR/HER3 antibody, CA1182, was generated from phage display library. CA1182 was as effective as cetuximab or trastuzumab in inhabiting phosphorylation of EGFR or HER2, but it exhibited as much more potent than cetuximab or trastuzumab. Moreover, our studies showed that CA1182 was significantly more effective than cetuximab in prolonging the survival of severe combined immune deficient mice bearing human NPC, suggesting that it might be a promising therapeutic agent for NPC.

Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.

Lung cancer has been the most prolific cancer in China – as in the rest of the world – with a high death rate and low 5-year survival rate. Previous evidence showed that JMJD2A is over-expressed in human non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues, and that high level of JMJD2A predicts poor overall and disease-free survival. However, the mechanism by which JMJD2A is regulated in human NSCLC is not fully understood. In the present study, we identified that the SIRT2 as an anti-oncogenic protein in NSCLC was down-regulated. JMJD2A as a target of SIRT2 was negatively correlated with SIRT2 level in NSCLC. SIRT2 bound to the promoter region of JMJD2A and negatively regulated JMJD2A expression. In addition, we found that SIRT2 inhibited NSCLC cells proliferation, colony formation and tumor growth in vitro and in vivo in a JMJD2A-dependent manner. In summary, our findings implicate that SIRT2 suppresses non-small cell lung cancer growth through targeting JMJD2A and SIRT2 activator may serve as candidate drug for NSCLC therapy.

Enhanced glutamine metabolism is required for tumor cell growth and survival, which suggests that agents targeting glutaminolysis may have utility within anti-cancer therapies. Troglitazone, a PPARγ agonist, exhibits significant anti-tumor activity and can alter glutamine metabolism in multiple cell types. Therefore, we examined whether troglitazone would disrupt glutamine metabolism in tumor cells and whether its action was reliant on PPARγ activity. We found that troglitazone treatment suppressed glutamine uptake and the expression of the glutamine transporter, ASCT2, and glutaminase. In addition, troglitazone reduced 13 C-glutamine incorporation into the TCA cycle, decreased [ATP], and resulted in an increase in reactive oxygen species (ROS). Further, troglitazone treatment decreased tumor cell growth, which was partially rescued with the addition of the TCA-intermediate, α-ketoglutarate, or the antioxidant N-acetylcysteine. Importantly, troglitazone’s effects on glutamine uptake or viable cell number were found to be PPARγ-independent. In contrast, troglitazone caused a decrease in c-Myc levels, while the proteasomal inhibitor, MG132, rescued c-Myc, ASCT2 and GLS1 expression, as well as glutamine uptake and cell number. Lastly, combinatorial treatment of troglitazone and metformin resulted in a synergistic decrease in cell number. Therefore, characterizing new anti-tumor properties of previously approved FDA therapies supports the potential for repurposing of these agents.