“Out of Pocket” Protein Binding—A Dilemma of Epitope Imprinted Polymers Revealed for Human Hemoglobin

The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.

LIN-35 beyond its classical roles: its function in the stress response

The pocket protein family controls several cellular functions such as cell cycle, differentiation, and apoptosis, among others; however, its role in stress has been poorly explored. The roundworm Caenorhabditis elegans is a simple model organism whose genes are highly conserved during evolution. C. elegans has only one pocket protein, LIN-35; a pRB-related protein similar to p130. To control the expression of some of its targets, LIN-35 interacts with E2F-DP transcription factors and LIN-52, a member of SynMUV (Synthetic Muv complex). Together, these proteins form the DRM complex, which is also known as the DREAM complex in mammals. In this review, we will focus on the role of LIN-35 and its partners in the stress response. It has been shown that LIN-35 is required to control starvation in L1 and L4 larval stages, and to induce starvation-induced germ apoptosis. Remarkably, during L1 starvation, insulin/IGF-1 receptor signaling (IIS), as well as the pathogenic, toxin, and oxidative stress-responsive genes, are repressed by LIN-35. The lack of lin-35 also triggers a downregulation of oxidative stress genes. Recent works showed that lin-35 and hpl-2 mutant animals showed enhanced resistance to UPRER. Additionally, hpl-2 mutant animals also exhibited the upregulation of autophagic genes, suggesting that the SynMuv/DRM proteins participate in this process. Finally, lin-35(n745) mutant animals overexpressed hsp-6, a chaperone that participated in the UPRmt. All of these data demonstrate that LIN-35 and its partners play an important role during the stress response.

DREAM Interrupted: Severing MuvB from DREAM’s pocket protein inCaenorhabditis elegansimpairs gene repression but not DREAM chromatin assembly

The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in mammalian systems, p107 and p130 represent the ancestral pocket proteins. The Rb-like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an E2F-DP transcription factor heterodimer, forming the DREAM (forDp,Rb-like,E2F,andMuvB) complex. DREAM complex formation on chromatin culminates in direct repression of target genes mediated by the MuvB subcomplex. Here, we examined how the Rb-like pocket protein contributes to DREAM formation by disrupting the interaction between the soleCaenorhabditis eleganspocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. Disrupting the LIN-35-MuvB association did not affect DREAM chromatin occupancy but did cause a highly penetrant synthetic multivulval (SynMuv) phenotype, indicating that blocking DREAM assembly impairs MuvB function. Some DREAM target genes became derepressed, indicating that for those genes MuvB chromatin binding alone is not sufficient for gene repression and that direct LIN-35-MuvB association potentiates MuvB’s innate repressive activity. In a previous study we showed that in worms lacking LIN-35, E2F-DP and MuvB chromatin occupancy is reduced genome-wide. With LIN-35 present, this study demonstrates that the E2F-DP-LIN-35 interaction promotes E2F-DP’s chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket protein family may recruit regulatory factors like MuvB to chromatin through E2F-DP to facilitate their transcriptional activity.

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.