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FRIMEDBIO-Fri prosj.st. med.,helse,biol

KARMA - an innovative method to analyze cellular fate of proteins and its application to probe the control of proteostasis

Alternative title: KARMA - en innovativ metode for å analysere cellulær skjebne til proteiner og dens anvendelse til å undersøke kontrollen av proteostase

Awarded: NOK 12.0 mill.

Cells produce numerous proteins and protein complexes that together conduct cell's life-sustaining activities. For example, ribosomes that make all cellular proteins or the mitochondrial respiratory chain that enables our cells to breathe. Yet, we know very little about how it is ensured that numerous cellular protein machineries remain functional. This question is not only of an academic value, but also has important medical implications. Well-heard-of ageing-related illnesses such as Alzheimer’s disease, Parkinson disease or diabetes are the consequence of inability to maintain proteostasis – a tendency for keeping the functional state of cell proteins. This leads to accumulation of non-functional and toxic protein aggregates and, ultimately, to cell death and disease. In this project we aim to scientifically understand the mechanisms of proteostasis and the consequences of its failure using innovative method, Kinetic Analysis of incorporation Rates in Macromolecular Assemblies, or, in short, KARMA. KARMA works on a similar basis as geological fossil dating. It uses isotopes (atoms with altered weight) to track fate of cellular proteins starting from their production to becoming part of the complexes and to the final elimination. Using KARMA, we focus on how proteostasis vulnerabilities can lead to disease. Firstly, we use KARMA to investigate the lifecycle of key protein machineries implicated in aging-related disorders. Secondly, we explore the impact of the proteostasis network malfunction – a specialized cellular machinery that ensures proteostasis – on the functional state cellular proteins. Thirdly, we investigate molecular mechanisms that lead to the development of health-treating protein aggregates observed in the proteostasis disorders, like in Alzheimer’s disease. To explore these aspects of proteostasis we use budding yeast, which is a very simple unicellular model organism. Despite simplicity, yeast have a Human-like proteostasis network and a highly similar list of protein machineries as implicated in the Human ageing-related disorders. The simplicity of yeast gives us a unique advantage of flexibly manipulating proteostasis and analyzing the consequences with KARMA. As for now KARMA methodology enabled us to describe lifecycle of the Nuclear Pore Complex, which is one of the most sophisticated disease-implicated protein assemblies, and discovering new elements that help cells produce these stuctures. Our already published results provided scientific community with a tool enabling to study different bio-generative processes such as assembly of protein complexes or for example viral reproduction (as in COVID). Our currently ongoing work improves the KARMA methodology, explores the impact of proteostasis network malfunction on the lifecycle of cellular proteins and on the development of non-functional protein aggregates.

To be properly functional, cells must keep myriad protein molecules and their complexes in a working condition, which is known as proteostasis. Loss of proteostasis is detrimental. For example, well heard-of illnesses including Alzheimer’s disease, Parkinson disease or diabetes originate from the cell's inability to keep the functional state of proteins. To understand the mechanisms of proteostasis and origins of its failure it is important to characterise the fate of cellular proteins (protein dynamics) including when and how they fold, assemble into multiprotein complexes, get modified and eliminated. Surprisingly, our knowledge of the protein dynamics is very fragmentary. For instance, the assembly mechanisms were studied for only a handful of ~4000 protein complexes present in mammals alone! To gain insight into the mechanisms of proteostasis, we propose to implement an innovative method entitled kinetic analysis of incorporation rates in macromolecular assemblies (KARMA), which would allow us to elucidate protein dynamics in the context of live cells. Using KARMA and budding yeast - a very powerful model system - we will address challenging questions in the field of proteostasis: How does the proteostasis network – the guardian of cellular proteins - keeps itself functional? How are vital cellular structures affected by the proteostasis failure? How exactly do disease-causing protein aggregates form in live cells? The implementation of KARMA requires an interdisciplinary effort at the intersection of biochemistry, quantitative mass-spectrometry and analysis of metabolic processes. KARMA is a very generic technique that can be used in various organisms not only to tackle the control of proteostasis but also to answer questions ranging from the formation of protein complexes to the mechanisms of viral infection.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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