Biology subjects

Tammsalu, T.

Publications and source records attributed to Tammsalu, T..

3 recordsLinked to original sources

bioRxiv2019

SUMOylation targets shugoshin to stabilize sister kinetochore biorientation

The accurate segregation of chromosomes during mitosis relies on the attachment of sister chromatids to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule remains unclear. Here we identify SUMOylation as a mechanism that promotes anaphase onset upon biorientation. SUMO ligases modify the tension-sensing pericentromere-localized chromatin protein, shugoshin, to stabilize bioriented sister kinetochore-microtubule attachments. In the absence of SUMOylation, Aurora B kinase removal from kinetochores is delayed. Shugoshin SUMOylation prevents its binding to protein phosphatase 2A (PP2A) and release of this interaction is important for stabilizing sister kinetochore biorientation. We propose that SUMOylation modulates the kinase-phosphatase balance within pericentromeres to inactivate the error correction machinery, thereby allowing anaphase entry in response to biorientation.

cell biology↗

bioRxiv2019

Controlling and co-ordinating chitinase secretion in a Serratia marcescens population

Serratia marcescens is a {gamma}-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein secretome comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the chitinolytic machinery, which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon and nitrogen source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, since normally only 1% of the population expresses the chitinolytic machinery. In this work, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60%. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.\n\nIMPORTANCEThe opportunistic pathogen Serratia marcescens secretes chitinases through the action of the chiWXYZ operon, which encodes a holin/endopeptidase pair. Expression of chiWXYZ is normally bimodal, with only 1% of the population transcribing these genes. In this work, it is demonstrated that overexpression of chiR induces exquisitely co-ordinated holin/endopeptidase and chitinase gene expression in the majority of the population of cells, establishing that ChiR is a key player in biomodal gene expression. This work also reveals for the first time that co-operating pathways are induced by ChiR, including enzymes involved in ammonia, nitrite and nitrate metabolism. This work expands knowledge of basic bacterial physiology and could have applications in the biomedical and biotechnological research fields.

microbiology↗

bioRxiv2017

Characterisation Of The Biflavonoid Hinokiflavone As A Pre-mRNA Splicing Modulator That Inhibits SENP

Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of purified E. coli expressed SUMO protease, SENP1 in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

biochemistry↗