MSc Biomedical and Molecular Sciences

BS52008 MSc Project Proposal and Associated Skills

Seminar Report

Name: Matriculation Number:

Email address:

Seminar title, presenter and affiliation:

Summary (30%)
[Describe the broad subject area of the presentation and summarize the key conclusions drawn. For seminars that present a series
of experimental studies, you should briefly describe the background to the work and summarize key aims or hypotheses that were
under investigation. The general experimental approach should be summarized (including, for example, animal models, analytical
techniques or data analysis methods) and key results should be provided, making sure that you explain how these relate to the
stated aims or hypotheses. Convey whether any of these results have changed the research direction of the field. You should limit
the length of this section to 500 words]
Write your answer here:

Using protein biochemistry and proteomics, the major signaling channels that control activation-
induced proteome remodeling in B- and T-cells were isolated and characterized. Insights on
Proteome remodeling in response to antigen and mammalian target of rapamycin complex
(mTORC1) were provided via quantitative mass spectrometry of CD4+ and CD8+ T cells.Naive T-
cells are obtained from mouse or human model system, and via in vitro stimulation they are
activated and they further differentiate into effector cells. Cells obtained from these three stages
are homogenized to yield peptides which are introduced to mass spectrometry, the output
determined the identity of their proteins. Inference from the results was used in the proteomics of
these cells. We showed that PCDC4 protein binds to EIF4A proteins in a 1:2 pattern to maintain the
quiescent state in T-cells, but this ratio roughly shifts to 1:55 within 24 hours of activation. Our
data also shows that mTORC1 inhibition has both similar and different effects on naive and
effector T cells. For example, while mTORC1 inhibition slowed down the cell cycle in activated
naive cells but had no effect on effector cells, it had a uniformly negative effect on the metabolism
of both T and B cells. By cross-examining the textbook perspective of mTORC1 activity and
providing a map of naive and effector T cell proteomes, this work facilitates the exploration and
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understanding of T cell phenotypes and cell context-specific functions of mTORC1. This study
demonstrates that immunological stimulation also causes down-regulation of a significant section
of the proteome in T cells. As a result, the Cantrell laboratory is shifting its focus to figuring out
how to regulate the dynamic cascade of protein breakdown that occurs during T-cell activation.
B cells play a critical role in the adaptive immune system by binding antigens through their
receptors, differentiating into effector plasma cells and memory populations, and secreting
cytokines, antibodies, and presenting antigens to T cells. Immunodeficiency, autoimmunity, and B
cell malignancies like leukemia and lymphoma are all linked to dysfunctional B cells. B cell
activation and differentiation, mostly driven by the transcriptional factor T-BET (which was
prioritized in the assay), and the distinctions between marginal zone and follicular B cells,
were studied with the help of knowledge gained in proteomics experts. Observations showed
that d differential protein landscapes explains why marginal zone B cells are quick to respond to
stimulation and follicular B cells take longer to do so. Toll-like receptors and other receptors
involved in environmental sensing and nutrient transport are highly expressed in marginal zone B
cells but not in follicular B cells. Follicular B cells, on the other hand, are what give rise to plasma
cells, which have a long lifespan and can access previously stored memories. For a healthy immune
system, it is therefore crucial to understand the protein landscape and how B-cells modulate their
protein landscape during development, activation, and differentiation. Understanding how the
plasma cell protein landscape is changing and how that may contribute to illness is also a driving
concern in the search to understand the influence of individual and combination stimuli on the B
cell phenotype during activation and differentiation using proteomics.

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Graphical Abstract (20%)
[Provide a self-generated graphical abstract (that does not mean it has to be sketched by hand; do not use online drawing sites or
icons/images obtained from such sites) summarising the principal conclusions of the seminar. Your abstract should be self-
contained, easily interpreted and should convey the context, primary mechanisms, and findings that were presented in the seminar.
Your abstract can be constructed using any graphic drawing programme (e.g. PowerPoint) and should be dropped into the box
below as a graphic image file (GIF, TIFF, JPEG) or similar. You might find the guidance given in the following publisher’s link useful:
http://www.cell.com/pb/assets/raw/shared/figureguidelines/GA_guide.pdf]
Add your answer here:

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Provide a figure legend to explain your graphical abstract
[The graphical abstract should be largely self-explanatory and so you should be concise here and ensure that you define
abbreviations. If your graphical abstract is organized into panels (e.g. A, B, C etc.) explain what each segment shows and how they
relate to one another but remember to give an overall title to the legend. You should limit the length of your figure legend to no
more than 100 words].
Write your answer here:

1 => Naive T-cells obtained from a laboratory rat. Just a few number of glucose and amino acid
transporters. Cell generates little energy. Some protein levels (e.g KLF2, PDCD4, IRF8) are high, to
keep the cell at quiescent state, while others such as PHD2, perforin, GranzymeB etc. are low.

2 => Stimulated T-cells gets activated. Up-regulation of nutrient transporters, specific proteomes
and down-regulation of other genes coding for KLF2, PDCD4 etc. Increased amount of ribosome,
mitochondria replicates and provides more energy. G1/S phase of cell cycle.

3 => Effector/ Differentiated T-Cells. T cells replicates rapidly, increased uptake of nutrient and
synthesis of translational machinery.

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Impact and implications (20%)
[Explain the value of this research, outlining the benefits (scientific, economic, societal, etc.) it is likely to bring to the wider scientific
community and/or to the global community. Explain how this research aligns to the mission statement and research goals of
relevant funding organization(s). You should limit the length of this section to 200 words.]
Write your answer here:

The research on proteome remodeling in B- and T-cells using protein biochemistry methods and
quantitative mass spectrometry has significant benefits for the scientific community, economy,
and society.

Scientifically, this research provides insights into the major signaling channels that control
proteome remodeling in response to antigen and mTORC1, shedding light on the metabolic and
protein production machinery and environmental sensors that determine T and B cell fate. The
identification and quantification of the selective proteome remodeling impact of activation using
proteomics and data from mass spectrometry provides a novel understanding of T and B cell
phenotypes. This research also facilitates the exploration and understanding of T and B cell
phenotypes and the cell context-specific functions of mTORC1. Furthermore, it provides insights
into the protein landscape of B cells during development, activation, and differentiation, which is
essential for a healthy immune system and in understanding how plasma cell protein landscape
changes and how that may contribute to illness.

Economically, this research can contribute to the development of new drugs and therapies for
immunodeficiency, autoimmunity, and B cell malignancies like leukemia and lymphoma.
Additionally, the findings can be used to develop new tools for diagnosis and treatment, improving
patient outcomes.

Socially, this research has the potential to benefit individuals with immune-related disorders and
diseases by leading to the development of more effective and targeted therapies. Furthermore,
understanding the immune system and how it responds to stimuli is crucial for the development of
vaccines and the prevention of infectious diseases.

This research aligns with the mission statement and research goals of funding organizations that
support research on immunology, proteomics, and cell biology. For example, the National
Institutes of Health (NIH) aims to advance knowledge and understanding of the immune system to
improve human health. Similarly, the European Union’s Horizon 2020 research and innovation
program seeks to support research on proteomics and molecular cell biology to develop new
therapies and improve human health. This research also aligns with the goals of organizations such
as the American Association of Immunologists, whose mission is to advance the field of
immunology to benefit human health.

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Q and A session and reflection (15%)
[Reflect on the questions after the seminar and resulting discussions in the context of your own knowledge and of the general
public. If no or few questions were asked, include your own questions/thoughts. You should limit the length of your answer to 200
words]
Write your answer here:

Question

I'm curious as to how protein turnover and degradation during T-cell activation and
differentiation relates to other processes, such as gene expression during transcription versus
protein turnover and translation. Are there similar studies looking at the transcriptome to try to
figure out how transcription plays a role in this regulatory process?

Answer
There have been parallel studies investigating the regulation of gene expression at both the
transcriptional and translational levels to address the question of how protein turnover and
degradation during T-cell activation and differentiation correlate with gene expression. It is well-
known that in many cell systems, T-cells included, there is a weak relationship between transcript
and protein levels. Since proteomics can reveal details about protein expression and regulation
that gene expression analysis cannot, it is an invaluable tool for this study.
Cyclin D3 is an example of a protein whose expression changes in response to transcriptional
regulation, but many other proteins, including PCDC4 and P27, undergo dramatic post-
transcriptional modifications. Current research is aimed at better understanding the mechanisms
that control the degradation and targeting of these proteins. It is well established that the
proteasome plays a role in controlling protein degradation, but the mechanisms by which
individual proteins are selected for degradation remain poorly understood. The molecular
mechanisms underlying T-cell activation and differentiation can be better understood by
examining both the transcriptome and the proteome changes.

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Key references (15%)
[Provide 3 peer-reviewed scientific publications that are relevant to the seminar material (no more than one from the presenter’s
lab and no review articles); provide for each a one sentence justification for selecting the particular reference. The references should
be presented in a suitable format (Vancouver or Harvard) with no errors or omission.]
Write your answer here:

Guo, Z., Primeau, T., Luo, J., Zhang, C., Sun, H., Hoog, J., Gao, F., Huang, S., Edwards, D.P., Davies,
S.R., Aft, R., Ding, L., Ellis, M.J., Li, S. and Ma, C.X. (2020). Proteomic Resistance Biomarkers for PI3K
Inhibitor in Triple Negative Breast Cancer Patient-Derived Xenograft Models. Cancers, [online]
12(12), p.3857. doi:https://doi.org/10.3390/cancers12123857.

This paper describes the use of proteomics to study the resistance mechanisms to PI3K inhibition
in triple negative breast cancers, which is similar to the research material in question.

Subbannayya, Y., Haug, M., Pinto, S.M., Mohanty, V., Meås, H.Z., Flo, T.H., Prasad, T.S.K. and
Kandasamy, R.K. (2020). The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal
Insights into Cell Differentiation and Function. International Journal of Molecular Sciences, 22(1),
p.275. doi:https://doi.org/10.3390/ijms22010275.

This study explores changes in the proteome of peripheral T cells in patients with Parkinson's
disease, which is similar to the research material that investigates changes in the proteome of T
cells during activation and differentiation.

Zhang, H., Yi, E.C., Li, X., Mallick, P., Kelly-Spratt, K.S., Masselon, C.D., Camp, D.G., Smith, R.D.,
Kemp, C.J. and Aebersold, R. (2005). High Throughput Quantitative Analysis of Serum Proteins
Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry. Molecular & Cellular
Proteomics, 4(2), pp.144–155. doi:https://doi.org/10.1074/mcp.m400090-mcp200.

This publication demonstrates the use of high-throughput quantitative analysis of serum proteins,
which is also a technique used in the research material to study the proteome of T cells.

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END