Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve exploitation of multiple evolutionarily conserved cellular signaling pathways including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/non-canonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands.

Systematic Modeling, Prediction, and Comparison of Domain–Peptide Affinities: Does it Work Effectively With the Peptide QSAR Methodology?

The protein–protein association in cellular signaling networks (CSNs) often acts as weak, transient, and reversible domain–peptide interaction (DPI), in which a flexible peptide segment on the surface of one protein is recognized and bound by a rigid peptide-recognition domain from another. Reliable modeling and accurate prediction of DPI binding affinities would help to ascertain the diverse biological events involved in CSNs and benefit our understanding of various biological implications underlying DPIs. Traditionally, peptide quantitative structure-activity relationship (pQSAR) has been widely used to model and predict the biological activity of oligopeptides, which employs amino acid descriptors (AADs) to characterize peptide structures at sequence level and then statistically correlate the resulting descriptor vector with observed activity data via regression. However, the QSAR has not yet been widely applied to treat the direct binding behavior of large-scale peptide ligands to their protein receptors. In this work, we attempted to clarify whether the pQSAR methodology can work effectively for modeling and predicting DPI affinities in a high-throughput manner? Over twenty thousand short linear motif (SLiM)-containing peptide segments involved in SH3, PDZ and 14-3-3 domain-medicated CSNs were compiled to define a comprehensive sequence-based data set of DPI affinities, which were represented by the Boehringer light units (BLUs) derived from previous arbitrary light intensity assays following SPOT peptide synthesis. Four sophisticated MLMs (MLMs) were then utilized to perform pQSAR modeling on the set described with different AADs to systematically create a variety of linear and nonlinear predictors, and then verified by rigorous statistical test. It is revealed that the genome-wide DPI events can only be modeled qualitatively or semiquantitatively with traditional pQSAR strategy due to the intrinsic disorder of peptide conformation and the potential interplay between different peptide residues. In addition, the arbitrary BLUs used to characterize DPI affinity values were measured via an indirect approach, which may not very reliable and may involve strong noise, thus leading to a considerable bias in the modeling. The Rprd2 = 0.7 can be considered as the upper limit of external generalization ability of the pQSAR methodology working on large-scale DPI affinity data.

The formation of a fuzzy complex in the negative arm regulates the robustness of the circadian clock.

The circadian clock times cellular processes to the day/night cycle via a Transcription-Translation negative Feedback Loop (TTFL). However, a mechanistic understanding of the negative arm in both the timing of the TTFL and its control of output is lacking. We posited that the formation of negative-arm protein complexes was fundamental to clock regulation stemming from the negative arm. Using a modified peptide microarray approach termed Linear motif discovery using rational design (LOCATE), we characterized the interaction of the disordered negative-arm clock protein FREQUENCY to its partner protein FREQUENCY-Interacting RNA helicase. LOCATE identified a specific Short Linear Motif (SLiM) and interaction hotspot as well as positively charged islands that mediate electrostatic interactions, suggesting a model where negative arm proteins form a fuzzy complex essential for clock timing and robustness. Further analysis revealed that the positively charged islands were an evolutionarily conserved feature in higher eukaryotes and contributed to proper clock function.

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both cohesin and condensin must be tightly regulated to adjust the structure of chromatids for their successful segregation.

RNF219 attenuates global mRNA decay through inhibition of CCR4-NOT complex-mediated deadenylation

The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.

Sensitive identification of known and unknown protease activities by unsupervised linear motif deconvolution

The cleavage-site specificities for many proteases are not well-understood, restricting the utility of supervised classification methods. We present an algorithm and web interface to overcome this limitation through the unsupervised detection of overrepresented patterns in protein sequence data, providing insight into the mixture of protease activities contributing to a complex system. Here, we apply the RObust LInear Motif Deconvolution (RoLiM) algorithm to confidently detect substrate cleavage patterns for SARS-CoV-2 Mpro protease in N terminome data of an infected human cell line. Using mass spectrometry-based peptide data from a case-control comparison of 341 primary urothelial bladder cancer cases and 110 controls, we identified distinct sequence motifs indicative of increased MMP activity in urine from cancer patients. Evaluation of N terminal peptides from patient plasma post-chemotherapy detected novel Granzyme B/Corin activity. RoLiM will enhance unbiased investigation of peptide sequences to establish the composition of known and uncharacterized protease activities in biological systems.

Robust unsupervised deconvolution of linear motifs characterizes 68 protein modifications at proteome scale

The local sequence context is the most fundamental feature determining the post-translational modification (PTM) of proteins. Recent technological improvements allow for the detection of new and less prevalent modifications. We found that established state-of-the-art algorithms for the detection of PTM motifs in complex datasets failed to keep up with this technological development and are no longer robust. To overcome this limitation, we developed RoLiM, a new linear motif deconvolution algorithm and webserver, that enables robust and unbiased identification of local amino acid sequence determinants in complex biological systems demonstrated here by the analysis of 68 modifications found across 30 tissues in the human draft proteome map. Furthermore, RoLiM analysis of a large-scale phosphorylation dataset comprising 30 kinase inhibitors of 10 protein kinases in the EGF signalling pathway identified prospective substrate motifs for PI3K and EGFR.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine Protein Phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R'. Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55α mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α role in multiple cellular processes.