Complex Insight - Understanding our world

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called “quantitative temporal viromics” (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

Via burkesquires

Bacteria adjust to different environmental conditions mainly by modulating transcription. Internal and external stimuli affect regulatory elements, all of which formulate the complex transcriptional profile of an organism at any given moment. In synthetic biology, the use of synthetic genetic circuits and metabolic pathways, which are often unresponsive to the aforementioned native regulation, is a common procedure. Such behavior can be advantageous, as it allows the organism to remain unaffected by unpredictable perturbations. In many cases, however, the lack of interaction with the internal control leads to undesired effects: metabolic intermediate accumulation, reduced fitness, and decreased product yields. This sets the framework of a recent research paper from the groups of Stephen Mayo and Richard Murray, where they describe a de novo transcription factor that is regulated by vanillin.

Vanillin is a byproduct of lignin degradation and an important substrate for the flavor industry. It is a phenolic compound with cytotoxic effects. In this article, the researchers modified qacR, a tetR-family repressor to bind vanillin, which binds to DNA via a helix-turn-helix domain. In the absence of the effector molecule, qacR physically inhibits RNA polymerase from transcribing the region downstream the binding site (see figure). The inducer causes conformational changes that prevent this binding, thus activating the gene. The procedure for qacR engineering consists of three steps:

(i) computational protein design. The researchers superimposed vanillin with the qacR crystal structure, enabling them to identify the potential binding conformations and the crucial aminoacids. They subsequently came up with a number of protein mutants that could form the correct interactions with vanillin and did not have steric clashes.

(ii) cell-free initial screening. The proteins resulting from the previous step were tested in an in vitro transcription/translation system. This methodology let the authors of the paper validate the repression qacR imposes on a reporter gene (GFP), while screening the functionality of the engineered proteins. Since all of the initial modified proteins failed to be activated by vanillin, the researchers went back to step (i) and designed more modifications. This time, two mutants showed the desired phenotype.

(iii) in vivo validation. As a last step, the two proteins from step (ii) were tested in E.coli. Both were able to suppress GFP expression in the lack of activator. One of them responded positively to increasing concentrations of vanillin, resulting in increased fluorescence.

Via Gerd Moe-Behrens

Cells regulate the uptake of nutrients and messenger cargos and their transport within the cell. This process is known as endocytosis and membrane traffic. Different cargos dock onto substrate specific receptors on the cell membrane. Special proteins such as kinases, GTPases and coats, activate specific entry routes and trigger the uptake of the receptors into the cell. For their uptake, the receptors and docked cargos become enclosed by the cell membrane. In the next steps, the membrane invaginates and becomes constricted. The resulting vesicle is guided via several distinct stations, cellular organelles, to its final destination in the cell.

 

For her study, Dr. Prisca Liberali, senior scientist in the team of Professor Lucas Pelkmans, sequentially switched off 1200 human genes. Using automated high-throughput light microscopy and computer vision, she could monitor and compare 13 distinct transport paths involving distinct receptors and cellular organelles. Precise quantifications of thousands of single cells identified the genes required for the different transport routes. Surprisingly, sets of transport routes are co-regulated and coordinated in specific ways by different programs of regulatory control.

 

Subsequently, Dr. Liberali calculated the hierarchical order within the genetic network and thereby identified the regulatory topology of cellular transport. "The transport into the cell and within the cells proceeds analogously to the cargo transport within a city" describes the scientist. "Like in a city, the traffic on the routes within a cell and their intersections is tightly regulated by traffic lights and signs to guide the cargo flow."

 

Thanks to this unique quantitative map, the fine regulatory details of transport paths and processes within a cells could be mapped for the first time. Particularly the genes that encode for these traffic lights and switches are often de-regulated in disease. With this map, it is now possible to predict how this leads to traffic jams in the cells, causing the disease phenotype. Alternatively, since many drugs have been developed to target these traffic lights and switches, the map can be used to come up with possible drug combinations to target unwanted traffic, such as viruses, to the waste disposal system of the cell.

Via Dr. Stefan Gruenwald, burkesquires