Biochemical Journal

The STK32 kinases are a small subfamily of three uncharacterised serine/threonine kinases from the AGC kinase family whose functional role is so far unknown. Here, we analyse the consensus peptide sequence for STK32A phosphorylation, showing that STK32A is directed towards acidic substrate sequences and exhibits dual-specificity for serine/threonine and tyrosine residues. A crystal structure of STK32A reveals an overall structure typical of the AGC protein kinase family but with significant and unique features including an altered binding mode of the hydrophobic motif to the N-terminal lobe of the kinase domain, and a novel alpha-helix in between the turn motif and the hydrophobic motif. The crystal structure combined with phylogenetic analysis reveals the evolutionary conservation of the acidic substrate preference. In vitro binding assays demonstrated that the STK32 kinases bind significant numbers of clinically used kinase inhibitors.

KANK proteins link integrin adhesions to the cortical microtubule stabilising complex (CMSC) through interactions with the adhesion adaptor talin. However, how KANK proteins are regulated remains unclear. Here we show that the KN domain of KANK proteins contains separable regions that mediate talin binding and a conserved intramolecular interaction. Using fluorescence polarisation, NMR spectroscopy and structural analysis, we map an interaction between the N-terminal KN domain and the C-terminal ankyrin repeat domain and identify residues 60-68 of the KN domain as required for this intramolecular interaction. In contrast, the canonical LD motif within residues 30-60 mediates binding to talin. Deletion of residues 60-68 disrupts the intramolecular interaction while preserving talin binding, demonstrating that the KN domain contains distinct modules for talin engagement and intramolecular regulation. This regulatory architecture is conserved across the KANK family, although sequence variation modulates the strength of the intramolecular interaction. Together, these findings identify a modular organisation within the KANK KN domain that separates talin recognition from intramolecular regulation and is consistent with an autoinhibitory mechanism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/731086v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@142543eorg.highwire.dtl.DTLVardef@1a8e454org.highwire.dtl.DTLVardef@1266c93org.highwire.dtl.DTLVardef@1a25fe7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules.The LD motif (blue) mediates binding to the talin R7 domain, whereas residues 60-68 (yellow) are required for interaction with the C-terminal ankyrin repeat domain. An AlphaFold model is shown as a structural interpretation of the intramolecular KN-ankyrin repeat interaction identified in this study. C_FIG

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of disability and mortality worldwide and the World Health Organisation has listed it with the highest priority for the need of new therapies. P. aeruginosa strains that express ExoU are implicated in the worst clinical outcomes. ExoU is phospholipase that is secreted by P. aeruginosa directly into the cytoplasm of target host cells, where its catalytic activity, directed towards plasma membranes, causes rapid cell lysis. Inhibition of ExoU may be a novel strategy to combat acutely cytotoxic ExoU expressing P. aeruginosa infections. Using an in vitro phospholipase assay, we performed a high throughput screen to identify compounds that might be repurposed as therapeutic ExoU inhibitors. We discovered a panel of compounds that appeared to inhibit ExoU through distinct mechanisms. Compound C prevented ExoU membrane localisation in HEK293T cells and caused colocalization with lysosomes, whereas compound D prevented PIP2 dependent oligomerisation of ExoU in vitro suggestive of synergistic action. Indeed, the concentrations required by compounds C and D to inhibit in vitro ExoU catalytic activity, when used in combination, was in the nanomolar region. In corneal scratch and infection assays, these compounds reduced ExoU mediated cytotoxicity, as assessed by fluorescence microscopy and lactate dehydrogenase release assays.

The Parkinsons Disease (PD)-linked protein Leucine Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lays within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM as well as the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

The ERM (ezrin, radixin and moesin) family of proteins and the related protein merlin participate in signaling events at the cell cortex. The proteins share an N-terminal FERM (band Four-point-one (4.1) ERM) domain comprised of three subdomains (F1, F2, and F3) that hold multiple binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that display peptides representing the intrinsically disordered regions of the human proteome we identified more than 220 FERM binding peptides. The majority of the peptides contained an apparent Yx[FILV] motif, but ligands with alternative motifs were also found. Interactions with thirteen peptides were validated using a fluorescence polarization assay, and interactions with seven full-length proteins were validated through pull-down experiments. We investigated the energy landscapes of interactions between the moesin FERM domain and representative set of ligands using Rosetta FlexPepDock computational peptide docking protocols, which provide a detailed molecular understanding of the binding of peptides with distinct motifs (YxV and E[Y/F]xDFYDF) to different sites on the F3 subdomain. A third motif (FY[D/E]L(4-5x)PLxxx[L/V]) was proposed to bind more diffusely. By combining competition and modeling experiments, we further uncovered interdependencies between different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin, and suggests that the FERM domain acts as a switchable interaction hub where one class of ligands to the F3 subdomain allosterically regulates binding of other F3 ligands.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 M. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa.

Remdesivir and Molnupiravir have gained considerable interest due to their activity against SARS-CoV-2. Cellular hydrolysis of their active triphosphate forms, Remdesivir-TP and Molnupiravir-TP, would decrease drug efficiency. We therefore tested Remdesivir-TP as a substrate against a panel of human hydrolases and found that NUDT18 catalyzes the hydrolysis of Remdesivir-TP. The kcat value of NUDT18 for Remdesivir-TP was determined to 2.6 s-1 and the Km value was 156 M, suggesting that NUDT18 catalyzed hydrolysis of Remdesivir-TP occurs in cells. We demonstrate that the triphosphates of the antivirals Ribavirin and Molnupiravir are hydrolyzed by NUDT18, albeit with a lower efficiency compared to Remdesivir-TP. NUDT18 also hydrolyses the triphosphates of Sofosbuvir and Aciclovir although with significantly lower activity. These results suggest that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of Remdesivir, Molnupiravir and Ribavirin. NUDT18 is expressed in respiratory epithelial cells and may limit the antiviral efficacy of Remdesivir and Molnupiravir against SARS-CoV2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action.

Toll-like and Interleukin-1/18 receptor resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using HDX-MS. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using 2pFLIM which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIREA protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Cyclic nucleotide second messengers are used in all domains of life to amplify viral infection signals and activate cellular defences. In prokaryotes, CBASS (cyclic nucleotide based antiphage signalling system) and type III CRISPR-Cas systems generate a range of cyclic nucleotides which bind and allosterically activate effector proteins to mount an anti-viral response. Viruses have evolved counter-measures to antagonise these signalling pathways in the form of cyclic nucleotide sponges and phosphodiesterases that sequester or degrade these molecules to subvert immunity. Recently, the Panoptes system was shown to function as a guard against these viral tactics. The type I Panoptes polymerase, mCpol, generates cyclic dinucleotides as decoy molecules that, when sequestered by phage proteins, results in the activation of the membrane-permeabilising effector 2TM{beta} to halt the phage infection cycle. Here, we investigate the type II Panoptes system, demonstrating that it generates cyclic tri-adenylate (cA3) to maintain a CRISPR-associated Rossmann fold-transmembrane (CARF-TM) effector in an inactive, dimeric state. When cA3 is sequestered or degraded, the CARF-TM protein oligomerises, resulting in increased outer membrane permeability and growth arrest. These findings expand our understanding of the guard systems that constitute a fascinating component of the bacterial immune system.

Biological engineers seek to have better control and a more complete understanding of the process of translation initiation within cells so that they may produce proteins more efficiently, as well as to create orthogonal translation systems. Previously, initiator tRNA variants have been created that initiate translation from non-AUG start codons, but their orthogonality has never been measured and the detailed characteristics of proteins produced from them have not been well defined. In this study we created an initiator tRNA mutant with anticodon altered to AAC to be complementary to GUU start codons. We deploy this i-tRNA(AAC) into E. coli cells and measure translation initiation efficiency against all possible start codons. Using parallel reaction monitoring targeted mass spectrometry we identify the N-terminal amino acids of i-tRNA(AAC)-initiated reporter proteins and show these proteins have altered stability within cells. We also use structural modeling of the peptide deformylase enzyme interaction with position 1 valine peptides to interrogate a potential mechanism for accumulation of formylated-valine proteins observed by mass spectrometry. Our results demonstrate that mutant initiator tRNAs have potential to initiate translation more orthogonally than the native initiator tRNA but their interactions with cellular formyltransferases and peptide deformylases can be inefficient because of the amino acid they are charged with. Additionally, engineered initiator tRNAs may enable tuning of in vivo protein stability through initiation with non-methionine amino acids that alter their interaction with cellular proteases.

Pseudomonas aeruginosa is one of the six bacteria of greatest concern identified by the WHO for its resistance to antibiotics. In the case of aminoglycosides, used in combination with other antibiotics in the treatment of severe infections, resistance is mainly due to modification of the antibiotic by bacterial enzymes. In this study, we have functionally and structurally characterized one such enzyme from P. aeruginosa, aminoglycoside 3-O-phosphotransferase IIb, APH(3)-IIb. The results will provide a better understanding of this resistance mechanism and enable us to envisage solutions for blocking it and restoring the efficacy of aminoglycosides.

The conditionally essential pathway of bacterial cysteine biosynthesis is gaining traction for the development of antibiotic adjuvants. Bacterial cysteine biosynthesis is generally facilitated by two enzymes possessing O-acetyl--serine sulfhydrylase (OASS) activity, CysK and CysM. CysK enzymes can also form functional complexes with other proteins that regulate cysteine metabolism. In Staphylococcus aureus there exists a single OASS homologue, herein termed SaCysK. Knockout of SaCysK was found to increase sensitivity to oxidative stress, making it a relevant target for inhibitor development. SaCysK forms two functional complexes via interaction with the preceding enzyme in the pathway serine acetyltransferase (CysE) or the transcriptional regulator of cysteine metabolism (CymR). These interactions occur through the insertion of a C-terminal peptide of CysE or CymR into the active site of SaCysK, inhibiting OASS activity, and therefore represent an excellent starting point for developing SaCysK inhibitors. Here we detail the characterization of CysE and CymR-derived C-terminal peptides as inhibitors of SaCysK. First, interactions between CysE or CymR-derived C-terminal decapeptides and SaCysK were assessed by X-ray crystallography. While both peptides occupied the active site of SaCysK, the alternate sidechains of the CymR decapeptide formed more extensive interactions. Surface plasmon resonance binding assays and SaCysK inhibition assays revealed that the CymR decapeptide bound to SaCysK with nanomolar affinity (KD = 25 nM) and inhibited SaCysK activity (IC50 = 180 nM), making it a promising lead for the development of SaCysK inhibitors. To understand the determinants of this high affinity interaction the structure-activity relationships of 16 rationally designed peptides were also investigated. This identified that the C-terminal pentapeptide of CymR alone facilitates the high affinity interaction with SaCysK, and that subtle structural modification of the pentapeptide is possible without impacting potency. Ultimately, this work has identified CymR pentapeptides as a promising scaffold for the development of antibiotic adjuvants targeting SaCysK. Author summaryThere is increasing interest in the investigation of non-essential pathways including bacterial cysteine metabolism for developing antibiotic adjuvants. Within this pathway the O-acetyl--serine sulfhydrylase (OASS) enzymes CysK and CysM have been a focus. As such, the OASS enzyme of Staphylococcus aureus, SaCysK, gained our interest. Previous efforts to inhibit CysK enzymes have mimicked the interaction between CysK and the C-terminus of serine acetyltransferase (CysE) which occurs inside the CysK active site and inhibits OASS activity. CysE peptides have only moderate potency, typically binding with micromolar affinity. In S. aureus another complex forms between SaCysK and a transcriptional regulator CymR, but the ability of CymR peptides to inhibit CysK enzymes has not been investigated. We noticed there is variation between the C-terminus of CysE and CymR, suggesting that CymR peptides make distinct interactions with SaCysK and may be superior inhibitors. Here we characterized CysE and CymR peptides as SaCysK inhibitors. We found CymR peptides make more extensive molecular interactions with SaCysK and bind with higher affinity, being the most potent peptide inhibitors of a CysK enzyme to date. A CymR pentapeptide is the minimal length required for this potency and provides a promising scaffold for developing antibiotic adjuvants targeting SaCysK.

The ATP binding cassette (ABC) family of transporters move diverse small molecules across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The allosteric mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) presents an unresolved question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved motif called the peptide sensor in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the peptide sensor motif alter ATP hydrolysis rates as well as substrate release. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects likely destabilize a network between the peptide sensor motif and the Q-loop and X-loop, two known allosteric elements in the NBDs. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the peptide sensor motifs help coordinate the transition of TmrAB to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

Sulfur metabolism plays an important role in bacterial pathogenesis. Elucidation of differences in sulfur metabolism across bacterial pathogens furthers our understanding of host survival and offers opportunities to disrupt these pathways for new therapies. Withing bacteria sulfur metabolism converges at the synthesis of L-cysteine. One of the key mechanisms of obtaining sulfur for the synthesis of L-cysteine is the successive reduction of sulfate to sulfide via the sulfate reduction pathway. Accordingly, L-cysteine biosynthesis is a critical metabolic pathway for bacterial survival, particularly in pathogenic species such as Neisseria gonorrhoeae and Staphylococcus aureus, which lack the sulfate reduction pathway. O-acetylserine sulfhydrylase catalyses the second step of the two-step synthesis reaction, condensing sulfide or thiosulfate (in the case of OASS-A/CysK or OASS-B/CysM respectively) with O-acetylserine to synthesize cysteine. Here we investigate the enzymatic properties and functional characterization of O-acetylserine sulfhydrylase, from N. gonorrhoeae and S. aureus, with a focus on substrate specificity, kinetic parameters, and cysteine synthase complex (CSC) formation. Using small angle X-ray scattering and kinetic assays we demonstrate that both N. gonorrhoeae and S. aureus CysK enzymes utilise only sodium sulfide for the synthesis of cysteine, despite the lack of a sulfate reduction pathway (to generate sulfide) in these organisms. Both enzymes demonstrate a higher affinity for O-acetylserine (OAS) compared to sodium sulfide (Na2S). We also show that the two cysteine synthesis enzymes, CysE and CysK that traditionally form the cysteine synthase complex do not form a complex in N. gonorrhoeae. These findings highlight the functional divergence in sulfur metabolism strategies among bacteria lacking sulfate reduction and provide deeper insights into the adaptive mechanisms of N. gonorrhoeae and S. aureus in sulfur flux.

Cyanophycin is a bacterial biopolymer used for storage of fixed nitrogen. It is composed of a backbone of L-aspartate residues with L-arginines attached to each of their side chains. Cyanophycin is produced by cyanophycin synthetase 1 (CphA1) using Arg, Asp and ATP, and is degraded in two steps. First, cyanophycinase breaks down the backbone peptide bonds, releasing {beta}-Asp-Arg dipeptides. Then, these dipeptides are broken down into free Asp and Arg by enzymes with isoaspartyl dipeptidase activity. Two bacterial enzymes are known to possess promiscuous isoaspartyl dipeptidase activity: isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA). We performed a bioinformatic analysis to investigate whether genes for cyanophycin metabolism enzymes cluster together or are spread around the microbial genomes. Many genomes showed incomplete contingents of known cyanophycin metabolizing genes. Cyanophycin synthetase and cyanophycinase are usually clustered together when recognizable genes for each are found within a genome. Cyanophycinase and isoaspartyl dipeptidase genes typically cluster within genomes lacking cphA1. About one-third of genomes with genes for CphA1, cyanophycinase and IaaA show these genes clustered together, while the proportion is around one-sixth for CphA1, cyanophycinase and IadA. We used X-ray crystallography and biochemical studies to characterize an IadA and an IaaA from two such clusters. The enzymes retained their promiscuous nature, showing that being associated with cyanophycin-related genes did not make them specific for {beta}-Asp-Arg dipeptides derived from cyanophycin degradation.

Insulin-degrading enzyme (IDE, insulysin, insulinase) is a peptidase that hydrolyzes a number of bioactive peptides including insulin and the amyloid beta peptide, making it a promising therapeutic target for diabetes and Alzheimers disease. Aspects of its physiological role are still controversial, however. In an attempt to further define IDEs role in cells, we used co-immunoprecipitation experiments to identify potential IDE interacting proteins. The enzyme pyrroline-5-carboxylate reductase 1 (PYCR1) was found associated with IDE in three different cell lines, and the two proteins colocalize in HeLa cells. Purified PYCR1 activates IDE toward small peptide substrates, suggesting a modulatory role for the interaction in vivo. Modeling suggests that the unstructured N-terminal region of PYCR1 inserts into allosteric sites of IDE, contributing to the observed activation. Deleting this sequence alters, but does eliminate, the interaction between PYCR1 and IDE. Since pyrroline-5-carboxylate reductase 1 is a mitochondrial protein, we posit that their interaction could regulate a previously described mitochondrial pool of IDE, which may serve to degrade mitochondrial targeting sequences or amyloid beta peptide that localizes to that organelle.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 M inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of [&le;] 100 M. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing. SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

Clusterin (CLU) is a constitutively secreted mammalian chaperone that binds in extracellular body fluids to misfolded client proteins to neutralise their toxicity and mediate their safe disposal by cell uptake and intracellular degradation. However, the regions of CLU critical for its interactions with misfolded proteins remain still largely unknown. To identify binding sites, we expressed a panel of CLU deletion and alanine-stretch mutants in a recently developed mammalian expression system. Mutant CLU molecules lacking detectable structural aberrations were subjected to functional analyses to compare their abilities with that of wild type CLU to bind to misfolded proteins and to inhibit protein aggregation. These analyses implicated two regions in the flexible {beta}-chain C-terminal tail of CLU as being important in the interactions of the chaperone with misfolded proteins, including aggregating the Alzheimers amyloid {beta}-peptide (A{beta}). We then designed in silico sequence-specific single-domain camelid nanobodies to confirm the function of the two putative client protein binding sites. Based on our experimental results and in silico binding site predictions, we suggest that the potent ability of CLU to promiscuously interact with many different misfolded proteins, regardless of their size or structure, arises from the location of multiple client protein binding sites in its flexible tail region.

ATP-dependent proteases play key roles in bacterial protein quality control and regulation of cellular processes. ClpYQ and ClpXP are ATP-dependent proteases in the Gram-positive bacteria Bacillus subtilis. To date, no substrate proteins of B. subtilis ClpYQ have been characterized. The protease component encoded by clpQ is synthetically lethal with clpP and the two genes are non-essential individually, suggesting potentially redundant roles for ClpYQ and ClpXP. Previous quantitative proteomic data predicted that B. subtilis proteins DivIVA and Mbl, components of the divisome and elongasome respectively, are potential substrates of ClpYQ. The role of DivIVA and Mbl in cell division and elongation suggests a significant role of ClpYQ in regulating cell division through targeted degradation of key divisome and elongasome proteins. Here we confirm that DivIVA and Mbl are degraded by ClpYQ both in vitro and in vivo, and thus identify the first two substrates of ClpYQ in B. subtilis.

BackgroundKRAS mutations are present in up to 30% of lung adenocarcinoma cases and are associated with poor survival. No effective targeted therapy against KRAS is currently available, and novel strategies to counteract oncogenic KRAS signaling are needed.\n\nResultsWe used targeted proteomics to monitor abundance and site-specific phosphorylation in a network of over 150 upstream and downstream effectors of KRAS signaling in H358 cells (KRAS G12C). We compared patterns of protein regulation following sustained signaling blockade in the RAS/ERK module at two different levels, KRAS and MEK. Network-based analysis demonstrated complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Among 85 most regulated proteins in the network, only 12 proteins showed concordant regulation in response to signaling blockade at both KRAS and MEK levels, while the remainder were either specifically regulated in response to KRAS knockdown or MEK inhibition or showed orthogonal regulation in both conditions. Dephosphorylation of DNA methyltransferase 1 (DNMT1) at S714 was identified among the changes unique to KRAS knockdown, and here we elucidate the role of this phosphorylation in KRAS-dependent transcriptional silencing of tumor suppressor genes.\n\nConclusionsNetwork-based analysis of the Ras signaling has shown complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Our work illustrates a targeted proteomics approach to functional interrogation of complex signaling networks focused on identification of readily testable hypotheses. These methods are widely applicable to diverse questions in tumor biology and other signaling paradigms.