α-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin

α-Synemin contains a unique 312 amino acid insert near the end of its C-terminal tail. Therefore we set out to determine if the insert is a site of protein–protein interaction that regulates the sub-cellular localization of this large isoform of synemin. Yeast-two hybrid analysis indicated that this region is a binding site for the M10 region of titin. This was confirmed with GST pull-down assays. Co-immunoprecipitation of endogenous proteins indicated close association of the two proteins in vivo and immunostaining of cardiomyocytes demonstrated co-localization of the proteins at the M-band of the sarcomere.     

Probing the druggability of membrane-bound Rab5 by molecular dynamics simulations

Rab5 is a small GTPase and a key regulator in early endosomal trafficking. Rab5 and its effectors are involved in a large number of infectious diseases and certain types of cancer. We performed µs atomistic molecular dynamics simulations of inactive and active full-length Rab5 anchored to a complex model bilayer with composition of the early endosome membrane. Direct interactions between the Rab5?G domain and the bilayer were observed. We found two dominant nucleotide-dependent orientations characterised by a different accessibility of the switch regions. The “buried switch” orientation was mainly associated with inactive Rab5 accompanied with a rather extended structure of the hypervariable C-terminal region. Active Rab5 preferred an orientation in which the switch regions are accessible to effector proteins. These structural differences may provide an opportunity to selectively target one Rab5 state and lead to new approaches in the development of Rab5-specific therapies.     

Hidden Multivalency in Phosphatase Recruitment by a Disordered AKAP Scaffold

Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.     

Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220

The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610–623) preferentially interacts with RII, whereas site 2 (residues 1633–1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β.     

Potential for polychlorinated biphenyl biodegradation in sediments from Indiana Harbor and Ship Canal

Polychlorinated biphenyls (PCBs) are carcinogenic, persistent, and bioaccumulative contaminants that pose risks to human and environmental health. In this study, we evaluated the PCB biodegradation of sediments from Indiana Harbor and Ship Canal (IHSC), a PCB-contaminated site (average PCB concentration = 12,570 ng/g dw). PCB congener profiles and bacterial community structure in a core sediment sample (4.57 m long) were characterized. Analysis of vertical PCB congener profile patterns in sediment and pore water strongly suggests that in situ dechlorination occurred in sediments. However, 16S rRNA genes from putative PCB-dechlorinating Chloroflexi were relatively more abundant in upper 2 m sediments, as were genes indicative of aerobic biodegradation potential (i.e. biphenyl dioxygenase (bphA)). Characterization of the bacterial community by terminal restriction fragment length polymorphism and comparison of these with sediment and pore water PCB congener profiles with the Mantel test revealed a statistical correlation (p < 0.001). Sequences classified as Acinetobacter and Acidovorax were highly abundant in deep sediments. Overall, our results suggest that PCB dechlorination has already occurred, and that IHSC sediments have the potential for further aerobic and anaerobic PCB biodegradation.     

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis

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