Functional analysis of the S100B protein and its variant isoform - vS100B

Abstract

100B is one of the most important members of the S100 family of proteins. These proteins are involved in numerous biological activities. Clinical studies of S100B have found their over expression in many types of cancer and leukaemia. Of particular interest is the role of elevated S100B in melanoma, where it is used as a marker to monitor the progression of malignant melanoma. Increased expression of S100B has also been demonstrated in T-cell leukaemia and astrocytic tumours. Unpublished study has shown a new variant of S100B (vS100B) that is likely produced by differential RNA splicing. S100B protein and its variant (vS100B) share amino acid homology in the N-terminal half, but are divergent from each other over the C- terminal half of the predicted protein. The objective of this study is to better understand the biological role of these proteins. Published data, has demonstrated the widespread expression of S100B in yeast, and differences in its protein-protein interactions depending on the nature of the different isoforms present. These studies have shown high expression levels for S100B and its isoform (vS100B) in brain. Interestingly, vS100B appears to interact with RABAC, AGTRAP, TMEM159, MAL2 and LOC100288797 in contrast with cS100B which does not appear to interact with these proteins. The originally identified isoform of S100B (cS100B) has been shown to bind p53, causing conformational changes that inhibit p53-dependent transcription of target genes including hdm2, p21 and bcl2. Therefore, S100B can affect a range of functions including DNA repair, cell cycle arrest and apoptosis. cS100B and vS100B are over expressed by many, but not all, malignantly transformed cells, including leukaemia and lymphoma cells. Therefore, S100B is a particularly attractive target for study, both for the better understanding of the underlying biology and for the potential development of new therapeutic strategies.

Studies described in this thesis have employed the cloning of cS100B and vS100B in expression vectors in order to examine the biological consequences of elevated expression of each of the two isoforms of S100B in transfected target cells. These studies have demonstrated the toxicity of both the classic and variant isoforms of S100B in HEK293T cells. By contrast, the variant S100B isoform had no toxicity in K562 cells and stably transfected K562 cells were isolated. The absence of an anti- vS100B antibody prevented the direct demonstration of vS100B protein in these cells. However, the use of a histidine tagged vS100B provided evidence for the presence of this protein in the transfected K562 cells.

In subsequent studies, transcription profiling was employed as a strategy for the identification of target genes the transcription of which was altered in the vS100B transfected K562 cells. This data detected no alteration in the transcript levels of a number of previously identified targets of S100B regulation, such as BCL-2, BCL-2 Like-1, Cyclin-dependent kinase (CDK) inhibitor P21 and murine double minute 2 (MDM2). However, there was a reduction of 2-3-fold in the level of another S100B target gene, myristoylated alanine-rich C-Kinase substrate (MARCKS). Interestingly, this reduction was detectable in K562 cells that over expressed either of the two isoforms of S100B, despite the major differences in the c-terminal half of the variant. The transcription profiling studies also identified a two-fold increase in the steady- state levels of two other genes: SNORD14E and MIR4275.

The Ingenuity Pathway Analysis (IPA), was used to identify mechanisms that may be affected by the altered expression of S100B isoforms. However, the identification of target genes the expression of which are altered by the altered expression of S100B isoforms, and potential differences between the effects of the different isoforms of S100B, remain in need of much more detailed studies. Specifically, detailed analysis of changes in RNA transcription profiles in cells with increased or decreased expression of either of the two isoforms of S100B remain to be extremely important.
However, one conclusion of the current studies is that no significant differences in transcription profiles of the cS100B and vS100B transfected K562 cells could be identified. Despite this observation, there is clear differences in the cellular responses to the overexpression of the different S100B isoforms. Specifically, the toxicity of both cS100B isoforms in HEK293T cells, but no toxicity for vS100B in K562 cells. The mechanisms involved in these major differences between the effects induced by vS100B and cS100B remain to be identified.

Date of Award	1 Feb 2022
Original language	English
Awarding Institution	King's College London
Supervisor	Farzin Farzaneh (Supervisor)