Alona Keren-Paz

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.

Recurrent inactivating RASA2 mutations in melanoma

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less efficiently than AzI1

ODC (ornithine decarboxylase), the first enzyme in the polyamine biosynthesis pathway in mammalian cells, is a labile protein. ODC degradation is stimulated by Az (antizyme), a polyamine-induced protein, which in turn is regulated by an ODC-related protein termed AzI (Az inhibitor). Recently, another ODCp (ODC paralogue) was suggested to function as AzI, on the basis of its ability to increase ODC activity and inhibit Az-stimulated ODC degradation in vitro. We show in the present study that ODCp is indeed capable of negating Az functions, as reflected by its ability to increase ODC activity and polyamine uptake and by its ability to provide growth advantage in stably transfected cells. However, ODCp is less potent than AzI1 in stimulating ODC activity, polyamine uptake and growth rate. The superiority of AzI1 to ODCp in inhibiting the Az-stimulated ODC degradation is also demonstrated using an in vitro degradation assay. We show that the basis for the inferiority of ODCp as an AzI is its lower affinity towards Az (Az1 and Az3). Further, we show here that ODCp, like AzI, is degraded in a ubiquitin-dependent manner, in a reaction that does not require either interaction with Az or the integrity of its C-terminus. Interaction with Az actually stabilizes ODCp by interfering with its ubiquitination. This results in sequestration of Az into a stable complex with ODCp, which is the central feature contributing to the ability of ODCp to function as AzI.

Antizyme 3 inhibits polyamine uptake and ornithine decarboxylase (ODC) activity, but does not stimulate ODC degradation

Azs (antizymes) are small polyamine-induced proteins that function as feedback regulators of cellular polyamine homoeostasis. They bind to transient ODC (ornithine decarboxylase) monomeric subunits, resulting in inhibition of ODC activity and targeting ODC to ubiquitin-independent proteasomal degradation. Az3 is a mammalian Az isoform expressed exclusively in testicular germ cells and therefore considered as a potential regulator of polyamines during spermatogenesis. We show here that, unlike Az1 and Az2, which efficiently inhibit ODC activity and stimulate its proteasomal degradation, Az3 poorly inhibits ODC activity and fails to promote ODC degradation. Furthermore, Az3 actually stabilizes ODC, probably by protecting it from the effect of Az1. Its inhibitory effect is revealed only when it is present in excess compared with ODC. All three Azs efficiently inhibit the ubiquitin-dependent degradation of AzI (Az inhibitor) 1 and 2. Az3, similar to Az1 and Az2, efficiently inhibits polyamine uptake. The potential significance of the differential behaviour of Az3 is discussed.

YAP and the drug resistance highway.

Deciphering mechanisms of drug resistance is crucial to winning the battle against cancer. A new study points to an unexpected function of YAP in drug resistance and illuminates its potential role as a therapeutic target.

Antizyme Affects Cell Proliferation and Viability Solely through Regulating Cellular Polyamines

Background: Antizyme is a regulator of cell proliferation, inhibiting this process when overexpressed. Results: Antizyme overexpression does not attenuate cell proliferation and viability in cells whose polyamine supply is secured. Conclusion: Antizyme affects cell proliferation and viability only by modulating polyamine metabolism. Significance: This result emphasizes the functional relationship of antizyme to cellular polyamine metabolism. Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.

Antizyme inhibitor: a defective ornithine decarboxylase or a physiological regulator of polyamine biosynthesis and cellular proliferation

ODC (ornithine decarboxylase) is a central regulator of cellular polyamine synthesis. ODC is a highly regulated enzyme stimulated by a variety of growth-promoting stimuli. ODC overexpression leads to cellular transformation. Cellular ODC levels are determined at transcriptional and translational levels and by regulation of its degradation. Here we review the mechanism of ODC degradation with particular emphasis on AzI (antizyme inhibitor), an ODC homologous protein that appears as a central regulator of ODC stability, cellular polyamine homoeostasis and cellular proliferation.

Micro-CT X-ray imaging exposes structured diffusion barriers within biofilms

In nature, bacteria predominantly exist as highly structured biofilms, which are held together by extracellular polymeric substance and protect their residents from environmental insults, such as antibiotics. The mechanisms supporting this phenotypic resistance are poorly understood. Recently, we identified a new mechanism maintaining biofilms - an active production of calcite minerals. In this work, a high-resolution and robust µCT technique is used to study the mineralized areas within intact bacterial biofilms. µCT is a vital tool for visualizing bacterial communities that can provide insights into the relationship between bacterial biofilm structure and function. Our results imply that dense and structured calcium carbonate lamina forms a diffusion barrier sheltering the inner cell mass of the biofilm colony. Therefore, µCT can be employed in clinical settings to predict the permeability of the biofilms. It is demonstrated that chemical interference with urease, a key enzyme in biomineralization, inhibits the assembly of complex bacterial structures, prevents the formation of mineral diffusion barriers and increases biofilm permeability. Therefore, biomineralization enzymes emerge as novel therapeutic targets for highly resistant infections.

Architects of nature: growing buildings with bacterial biofilms

In his text ‘On Architecture’, Vitruvius suggested that architecture is an imitation of nature. Here we discuss what happens when we begin using nature in architecture. We describe recent developments in the study of biofilm structure, and propose combining modern architecture and synthetic microbiology to develop sustainable construction approaches. Recently, Kolodkin‐Gal laboratory and others revealed a role for precipitation of calcium carbonate in the maturation and assembly of bacterial communities with complex structures. Importantly, they demonstrated that different secreted organic materials shape the calcium carbonate crystals formed by the bacterial cells. This provides a proof‐of‐concept for a potential use of bacteria in designing rigid construction materials and altering crystal morphology and function. In this study, we discuss how these recent discoveries may change the current strategies of architecture and construction. We believe that biofilm communities enhanced by synthetic circuits may be used to construct buildings and to sequester carbon dioxide in the process.

Collective Vortex-Like Movement of Bacillus subtilis Facilitates the Generation of Floating Biofilms

Bacteria in nature are usually found in complex multicellular structures, called biofilms. One common form of a biofilm is pellicle—a floating mat of bacteria formed in the water-air interphase. So far, our knowledge on the basic mechanisms underlying the formation of biofilms at air-liquid interfaces is not complete. In particular, the co-occurrence of motile cells and extracellular matrix producers has not been studied. In addition, the potential involvement of chemical communication in pellicle formation remained largely undefined. Our results indicate that vortex-like collective motility by aggregates of motile cells and EPS producers accelerate the formation of floating biofilms. Successful aggregation and migration to the water-air interphase depend on the chemical communication signal autoinducer 2 (AI-2). This ability of bacteria to form a biofilm in a preferable niche ahead of their potential rivals would provide a fitness advantage in the context of inter-species competition.