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Isolation of isoform-specific binding proteins (Affimers) by phage display using negative selection


Some 30 years after its discovery, phage display remains one of the most widely used methods of in vitro selection. Initially developed to revolutionize the generation of therapeutic antibodies, phage display is now the first choice for screening artificial binding proteins. Artificial binding proteins can be used as reagents to study protein-protein interactions, target posttranslational modifications, and distinguish between homologous proteins. They can also be used as research and affinity reagents, for diagnostic purposes, and as therapeutics. However, the ability to identify isoform-specific reagents remains highly challenging. We describe an adapted phage display protocol using an artificial binding protein (Affimer) for the selection of isoform-selective binding proteins.

Generation of specific inhibitors of SUMO-1- and SUMO-2/3-mediated protein-protein interactions using Affimer (Adhiron) technology


Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.

The genetics of PKM{zeta} and memory maintenance


Elucidating the molecular mechanisms that maintain long-term memory is a fundamental goal of neuroscience. Accumulating evidence suggests that persistent signaling by the atypical protein kinase C (PKC) isoform protein kinase M (PKM) might maintain synaptic long-term potentiation (LTP) and long-term memory. However, the role of PKM has been challenged by genetic data from PKM-knockout mice showing intact LTP and long-term memory. Moreover, the PKM inhibitor peptide inhibitory peptide (ZIP) reverses LTP and erases memory in both wild-type and knockout mice. Data from four papers using additional isoform-specific genetic approaches have helped to reconcile these conflicting findings. First, a PKM-antisense approach showed that LTP and long-term memory in PKM-knockout mice are mediated through a compensatory mechanism that depends on another ZIP-sensitive atypical isoform, PKC/. Second, short hairpin RNAs decreasing the amounts of individual atypical isoforms without inducing compensation disrupted memory in different temporal phases. PKC/ knockdown disrupted short-term memory, whereas PKM knockdown specifically erased long-term memory. Third, conditional PKC/ knockout induced compensation by rapidly activating PKM to preserve short-term memory. Fourth, a dominant-negative approach in the model system Aplysia revealed that multiple PKCs form PKMs to sustain different types of long-term synaptic facilitation, with atypical PKM maintaining synaptic plasticity similar to LTP. Thus, under physiological conditions, PKM is the principal PKC isoform that maintains LTP and long-term memory. PKC/ can compensate for PKM, and because other isoforms could also maintain synaptic facilitation, there may be a hierarchy of compensatory mechanisms maintaining memory if PKM malfunctions.

Papers of note in Science Translational Medicine 9 (415)


This week’s articles describe a way to combat the negative effects of stress on cancer therapy; a potential target in heart failure; and why nighttime wounds heal more slowly than daytime wounds.

Papers of note in Science 358 (6364)


This week’s articles highlight a newly identified component of methionine sensing; a mechanism by which nutrient depletion prevents the efflux of essential amino acids from lysosomes; how reactive oxygen species promote genome stability during metabolic stress; co-option of the cell cycle regulatory machinery to time the differentiation of multiciliated cells; and connections between olfaction, organismal metabolism, and longevity.

Papers of note in Nature 551 (7679)


This week’s articles highlight interactions between astrocytes and neurons that influence both cell types’ form and function; a genetic basis for individual variation in aging; and the mechanism by which an E3 ubiquitin ligase adaptor affects synaptic transmission.

Fat expansion through norepinephrine catabolism


Certain macrophage populations limit norepinephrine-induced lipolysis in adipose tissues in response to aging or obesity.

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia


Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including MYC and HES1, and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.