Download IN THIS ISSUE Mutating it all Discovering ubiquitylation

NATURE METHODS | VOL.13 NO.11 | NOVEMBER 2016
IN THIS ISSUE
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
CRISPR rewires cell signaling
Cellular function depends on signaling networks
to process information and elicit a desired
outcome. This intracellular communication can
be modified with engineered molecules that
interrupt or redirect signals, but integrating
information from different signaling inputs
remains a challenge. Liu and colleagues created
signal conductors based on the CRISPR system:
an aptamer responding to a ligand was fused to
the guide RNA, and the conformational change
after ligand binding allowed the sgRNA to bind
Cas9 and trigger transcriptional activation or
repression, depending on the Cas9 molecule used.
The researchers reprogrammed cancer cells to
respond to an oncogenic stimulus by inducing
the expression of genes that trigger cell death.
Tumors in mice carrying these engineered cancer
cells were smaller than those in control animals.
Article p938
Bypassing cysteine in expressed
protein ligation
Expressed protein ligation (EPL) is a powerful tool for
synthetically generating proteins with desired posttranslational modifications. In EPL, one portion of a
target protein is expressed in cells as a fusion to an
intein, which cleaves itself off, leaving a C-terminal
thioester. This thioester can then be ligated to a
synthetic peptide bearing an N-terminal cysteine
along with modifications of interest. However, this
often introduces a non-native cysteine into the
protein of interest, which can affect its function. Cole
and colleagues developed an approach that uses the
subtiligase enzyme to ligate proteins with C-terminal
thioesters to peptides lacking N-terminal cysteines.
They demonstrate that subtiligase can catalyze
ligation with an array of peptides, and using the
protein PTEN they show that proteins with the native
sequence outperform those with non-native cysteines
in biological experiments.
Brief Communication p925
Monitoring zinc homeostasis
Zinc is an important metal in biological systems. Work
using fluorescent probes to detect Zn2+ has shown
that its distribution in cells can vary widely depending
on the conditions. However, many questions remain
about how zinc homeostasis is regulated in cells.
To identify proteins involved in zinc homeostasis,
Hamachi and colleagues used a ‘conditional
proteomics’ approach. They developed a proteinlabeling reagent, AIZin, that is activated only in the
presence of Zn2+. In areas of high Zn2+ concentration
in the cell, AIZin will label proximal proteins, which
can then be detected and identified using mass
spectrometry analysis. The authors applied the
method to study how zinc homeostasis is disrupted
under conditions of oxidative stress in glioma cells.
With the development of specific chemical labeling
reagents, conditional proteomics could be adapted to
study many different biological targets.
Article p931, News & Views p917
Mutating it all
Deep mutational scanning libraries are the starting
point for structure–function mapping, in vitro
evolution studies, enhancer assays, genetic analyses
and a host of other important applications. Two
papers in this issue make it easier to generate
large-scale mutation libraries in plasmids. Haller
and colleagues achieved massively parallel singlenucleotide mutagenesis by using reversibly terminated
deoxyinosine triphosphates, which are incorporated
randomly during linear amplification and ensure
a single mutation per molecule. Whitehead and
colleagues simplified primer-mediated PFunkel
mutagenesis into a single-day, one-pot protocol
by sequentially applying endonucleases that nick
opposite strands. Their nicking mutagenesis method
can generate comprehensive single- and multisite
saturation mutagenesis libraries with high efficiency,
or it can be used for more restricted mutagenesis.
Brief Communications p923, p928
Discovering ubiquitylation
cascades
Ubiquitylation is a crucial
regulator of almost
all eukaryotic cellular
pathways. This process
is complex, requiring the
concerted action of E1, E2
and E3 enzymes, as well as
deubiquitylases. Sorting
out which of the hundreds of enzymes are responsible
for acting on which target proteins has proved to
be quite challenging. Prag and colleagues report a
genetic selection system in Escherichia coli to help link
enzymes in the ubiquitylation cascade to their targets.
The system is based on a split DHFR reporter gene—
one reporter fragment is linked to ubiquitin, and the
other to the target protein; both are coexpressed along
with the ubiquitylation apparatus. If the target protein
is ubiquitylated, the DHFR reporter assembles into a
functional enzyme and confers antibiotic resistance.
The authors used the method to screen a yeast library,
discovering a novel substrate of the E3 Rsp5.
Article p945