Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Stay of execution

26.01.2009
A new twist on an old technique helps researchers identify proteins with a regulatory ‘death sentence’

The targeted destruction of specific proteins is an important means of regulation for many cellular pathways. This is typically managed through the process called ubiquitination, in which doomed proteins are chemically marked for entry into a degradation pathway by protein complexes known as ubiquitin ligases.

“Although the identification of substrates is essential for our understanding of cellular regulatory mechanisms involving ubiquitination, identifying them is quite difficult,” explains Tsutomu Kishi of the Advanced Science Institute in Wako, whose work on ubiquitin ligase target recognition via subunits known as ‘F-box proteins’ has been impeded by the limitations of existing tools for protein–protein interaction analysis.

One popular method is the ‘two-hybrid’ system, which uses a gene-activating protein that has been split into two pieces: one capable of binding to a target DNA sequence, and one capable of inducing activation. The first piece is fused to a ‘bait’ protein, while the second piece is fused to various ‘prey’ proteins; both bait and prey are then introduced into yeast cells with an indicator gene containing an appropriate binding site for the bait. The indicator is only turned on if the DNA-binding domain and gene activation domain become linked via prey–bait interaction, making it easy to identify such associations.

When working with ubiquitination targets, however, prey fusions are in danger of being marked for rapid destruction by the host cell before interactions can be detected. Kishi and colleagues therefore modified the assay so that it could be performed under conditions in which the relevant degradation pathways are disabled, enabling straightforward two-hybrid analysis of substrates from these pathways1.

Kishi’s team applied their method to Cdc4, a component of the SCFCdc4 ubiquitin ligase complex. They identified four interacting partners, but focused on Swi5, a protein that stimulates production of SIC1, a regulator that inhibits onset of S phase—and also a ubiquitination target. Subsequent experiments revealed that SCFCdc4 mediates a two-pronged process of SIC1 downregulation by first reducing levels of the activator protein Swi5, and then by inducing direct degradation of SIC1 itself.

These findings offer valuable insights into the regulation of the cell cycle and illustrate an important ‘indirect’ mechanism for ubiquitination-based regulation of protein levels via the targeting of relevant gene activators for destruction. They also demonstrate the effectiveness of a strategy that could be generalized for identifying other ubiquitination targets. “This methodology is widely applicable,” says Kishi, “and in collaboration with other groups, we have succeeded in identifying targets of other F-box proteins.”

Reference

1. Kishi, T., Ikeda, A., Koyama, N., Fukada, J. & Nagao, R. A refined two-hybrid system reveals that SCFCdc4-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry. Proceedings of the National Academy of Sciences USA 105, 14497–14502 (2008).

Saeko Okada | ResearchSEA
Further information:
http://www.rikenresearch.riken.jp/research/628/

More articles from Life Sciences:

nachricht Mass spectrometry sheds new light on thallium poisoning cold case
14.12.2018 | University of Maryland

nachricht Protein involved in nematode stress response identified
14.12.2018 | University of Illinois College of Agricultural, Consumer and Environmental Sciences

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Data use draining your battery? Tiny device to speed up memory while also saving power

The more objects we make "smart," from watches to entire buildings, the greater the need for these devices to store and retrieve massive amounts of data quickly without consuming too much power.

Millions of new memory cells could be part of a computer chip and provide that speed and energy savings, thanks to the discovery of a previously unobserved...

Im Focus: An energy-efficient way to stay warm: Sew high-tech heating patches to your clothes

Personal patches could reduce energy waste in buildings, Rutgers-led study says

What if, instead of turning up the thermostat, you could warm up with high-tech, flexible patches sewn into your clothes - while significantly reducing your...

Im Focus: Lethal combination: Drug cocktail turns off the juice to cancer cells

A widely used diabetes medication combined with an antihypertensive drug specifically inhibits tumor growth – this was discovered by researchers from the University of Basel’s Biozentrum two years ago. In a follow-up study, recently published in “Cell Reports”, the scientists report that this drug cocktail induces cancer cell death by switching off their energy supply.

The widely used anti-diabetes drug metformin not only reduces blood sugar but also has an anti-cancer effect. However, the metformin dose commonly used in the...

Im Focus: New Foldable Drone Flies through Narrow Holes in Rescue Missions

A research team from the University of Zurich has developed a new drone that can retract its propeller arms in flight and make itself small to fit through narrow gaps and holes. This is particularly useful when searching for victims of natural disasters.

Inspecting a damaged building after an earthquake or during a fire is exactly the kind of job that human rescuers would like drones to do for them. A flying...

Im Focus: Topological material switched off and on for the first time

Key advance for future topological transistors

Over the last decade, there has been much excitement about the discovery, recognised by the Nobel Prize in Physics only two years ago, that there are two types...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

ICTM Conference 2019: Digitization emerges as an engineering trend for turbomachinery construction

12.12.2018 | Event News

New Plastics Economy Investor Forum - Meeting Point for Innovations

10.12.2018 | Event News

EGU 2019 meeting: Media registration now open

06.12.2018 | Event News

 
Latest News

Data use draining your battery? Tiny device to speed up memory while also saving power

14.12.2018 | Power and Electrical Engineering

Tangled magnetic fields power cosmic particle accelerators

14.12.2018 | Physics and Astronomy

In search of missing worlds, Hubble finds a fast evaporating exoplanet

14.12.2018 | Physics and Astronomy

VideoLinks
Science & Research
Overview of more VideoLinks >>>