Structural Analysis of the Protein Phosphatase 1 Docking Motif: Molecular Description of Binding Specificities Identifies Interacting Proteins

SummaryThe interplay between kinases and phosphatases represents a fundamental regulatory mechanism in biological systems. Being less numerous than kinases, phosphatases increase their diversity by the acquisition of a variety of binding partners, thereby forming a large number of holoenzymes. Proteins interacting with protein phosphatase 1 (PP1) often bind via a so-called docking motif to regulate its enzymatic activity, substrate specificity, and subcellular localization. Here, we systematically determined structural elements that mediate the binding specificity of PP1 interacting proteins, and propose a refined consensus sequence for high-affinity PP1 ligands. Applying this pattern to database searches, we predicted and experimentally confirmed several previously unknown PP1 interactors. Thus, the suggested PP1 docking motif enables a highly specific prediction of PP1 binding partners, thereby facilitating the genome-wide identification of PP1 interactors

Development and Validation of a Prediction Model for Future Estimated Glomerular Filtration Rate in People With Type 2 Diabetes and Chronic Kidney Disease

Importance: Type 2 diabetes increases the risk of progressive diabetic kidney disease, but reliable prediction tools that can be used in clinical practice and aid in patients' understanding of disease progression are currently lacking. Objective: To develop and externally validate a model to predict future trajectories in estimated glomerular filtration rate (eGFR) in adults with type 2 diabetes and chronic kidney disease using data from 3 European multinational cohorts. Design, Setting, and Participants: This prognostic study used baseline and follow-up information collected between February 2010 and December 2019 from 3 prospective multinational cohort studies: PROVALID (Prospective Cohort Study in Patients with Type 2 Diabetes Mellitus for Validation of Biomarkers), GCKD (German Chronic Kidney Disease), and DIACORE (Diabetes Cohorte). A total of 4637 adult participants (aged 18-75 years) with type 2 diabetes and mildly to moderately impaired kidney function (baseline eGFR of ≥30 mL/min/1.73 m2) were included. Data were analyzed between June 30, 2021, and January 31, 2023. Main Outcomes and Measures: Thirteen variables readily available from routine clinical care visits (age, sex, body mass index; smoking status; hemoglobin A1c[mmol/mol and percentage]; hemoglobin, and serum cholesterol levels; mean arterial pressure, urinary albumin-creatinine ratio, and intake of glucose-lowering, blood-pressure lowering, or lipid-lowering medication) were selected as predictors. Repeated eGFR measurements at baseline and follow-up visits were used as the outcome. A linear mixed-effects model for repeated eGFR measurements at study entry up to the last recorded follow-up visit (up to 5 years after baseline) was fit and externally validated. Results: Among 4637 adults with type 2 diabetes and chronic kidney disease (mean [SD] age at baseline, 63.5 [9.1] years; 2680 men [57.8%]; all of White race), 3323 participants from the PROVALID and GCKD studies (mean [SD] age at baseline, 63.2 [9.3] years; 1864 men [56.1%]) were included in the model development cohort, and 1314 participants from the DIACORE study (mean [SD] age at baseline, 64.5 [8.3] years; 816 men [62.1%]) were included in the external validation cohort, with a mean (SD) follow-up of 5.0 (0.6) years. Updating the random coefficient estimates with baseline eGFR values yielded improved predictive performance, which was particularly evident in the visual inspection of the calibration curve (calibration slope at 5 years: 1.09; 95% CI, 1.04-1.15). The prediction model had good discrimination in the validation cohort, with the lowest C statistic at 5 years after baseline (0.79; 95% CI, 0.77-0.80). The model also had predictive accuracy, with an R2ranging from 0.70 (95% CI, 0.63-0.76) at year 1 to 0.58 (95% CI, 0.53-0.63) at year 5. Conclusions and Relevance: In this prognostic study, a reliable prediction model was developed and externally validated; the robust model was well calibrated and capable of predicting kidney function decline up to 5 years after baseline. The results and prediction model are publicly available in an accompanying web-based application, which may open the way for improved prediction of individual eGFR trajectories and disease progression.</p

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.This work was supported by the Deutsche Forschungsgemeinschaft (Grant DFG VI586 to C.V.) and the European Union (FP7 project Neurocypres to C.J.K., K.L.P., and S.C.R.L.). N. Schaefer and G.L. are supported by the GSLS Wuerzburg. S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Research.This is the author accepted manuscript. The final version is available from the Society of Neuroscience via http://dx.doi.org/10.1523/JNEUROSCI.1509-14.201

ELM—the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instance

Genetic studies of paired metabolomes reveal enzymatic and transport processes at the interface of plasma and urine.

The kidneys operate at the interface of plasma and urine by clearing molecular waste products while retaining valuable solutes. Genetic studies of paired plasma and urine metabolomes may identify underlying processes. We conducted genome-wide studies of 1,916 plasma and urine metabolites and detected 1,299 significant associations. Associations with 40% of implicated metabolites would have been missed by studying plasma alone. We detected urine-specific findings that provide information about metabolite reabsorption in the kidney, such as aquaporin (AQP)-7-mediated glycerol transport, and different metabolomic footprints of kidney-expressed proteins in plasma and urine that are consistent with their localization and function, including the transporters NaDC3 (SLC13A3) and ASBT (SLC10A2). Shared genetic determinants of 7,073 metabolite-disease combinations represent a resource to better understand metabolic diseases and revealed connections of dipeptidase 1 with circulating digestive enzymes and with hypertension. Extending genetic studies of the metabolome beyond plasma yields unique insights into processes at the interface of body compartments

Blood pressure control in chronic kidney disease: A cross-sectional analysis from the German Chronic Kidney Disease (GCKD) study

We assessed the prevalence, awareness, treatment and control of hypertension in patients with moderate chronic kidney disease (CKD) under nephrological care in Germany. In the German Chronic Kidney Disease (GCKD) study, 5217 patients under nephrology specialist care were enrolled from 2010 to 2012 in a prospective observational cohort study. Inclusion criteria were an estimated glomerular filtration rate (eGFR) of 30 +/- 60 mL/min/1.73 m 2 or overt proteinuria in the presence of an eGFR> 60 mL/min/1.73 m 2. Office blood pressure was measured by trained study personnel in a standardized way and hypertension awareness and medication were assessed during standardized interviews. Blood pressure was considered as controlled if systolic 90%. However, only 2456 (49.3%) of the hypertensive patients had controlled blood pressure. About half (51.0%) of the patients with uncontrolled blood pressure met criteria for resistant hypertension. Factors associated with better odds for controlled blood pressure in multivariate analyses included younger age, female sex, higher income, low or absent proteinuria, and use of certain classes of antihypertensive medication. We conclude that blood pressure control of CKD patients remains challenging even in the setting of nephrology specialist care, despite high rates of awareness and medication use

Differential prognostic utility of adiposity measures in chronic kidney disease

Objective Adipose tissue contributes to adverse outcomes in chronic kidney disease (CKD), but there is uncertainty regarding the prognostic relevance of different adiposity measures. We analyzed the associations of neck circumference (NC), waist circumference (WC), and body mass index (BMI) with clinical outcomes in patients with mild to severe CKD. Methods The German Chronic Kidney Disease (GCKD) study is a prospective cohort study, which enrolled Caucasian adults with mild to severe CKD, defined as estimated glomerular filtration rate (eGFR): 30–60 mL/min/1.73 m2, or >60 mL/min/1.73 m2 in the presence of overt proteinuria. Associations of NC, WC and BMI with all-cause death, major cardiovascular events (MACE: a composite of non-fatal stroke, non-fatal myocardial infarction, peripheral artery disease intervention, and cardiovascular death), kidney failure (a composite of dialysis or transplantation) were analyzed using multivariable Cox proportional hazards regression models adjusted for confounders and the Akaike information criteria (AIC) were calculated. Models included sex interactions with adiposity measures. Results A total of 4537 participants (59% male) were included in the analysis. During a 6.5-year follow-up, 339 participants died, 510 experienced MACE, and 341 developed kidney failure. In fully adjusted models, NC was associated with all-cause death in women (HR 1.080 per cm; 95% CI 1.009–1.155), but not in men. Irrespective of sex, WC was associated with all-cause death (HR 1.014 per cm; 95% CI 1.005–1.038). NC and WC showed no association with MACE or kidney failure. BMI was not associated with any of the analyzed outcomes. Models of all-cause death including WC offered the best (lowest) AIC. Conclusion In Caucasian patients with mild to severe CKD, higher NC (in women) and WC were significantly associated with increased risk of death from any cause, but BMI was not

Computational analysis of structure, dynamics, and energetics of protein interactions

Proteininteraktionen spielen praktisch bei allen biologischen Prozessen eine wesentliche Rolle. Um die zugrunde liegenden molekularen Vorgänge zu verstehen, ist es notwendig die Struktur und Dynamik von Proteinen auf atomarer Ebene zu charakterisieren. In der vorliegenden Arbeit wurde eine Reihe von Proteininteraktionen eingehend mit verschiedenen in silico und in vitro Methoden untersucht. Die Koordination von Abläufen in biologischen Systemen erfordert das Vorhandensein verschiedener Signalübertragungswege, die untereinander in Wechselwirkung stehen und exakt reguliert werden müssen. Die Tyrosinkinasen der Src-Familie, die am Anfang vieler Signalwege stehen unterscheiden sich trotz ihrer großen strukturellen Ähnlichkeit in ihren physiologischen Funktionen. In früheren Arbeiten wurde bereits gezeigt, dass die Flexibilität des SH3-SH2 Domänenlinkers kritisch für die relative Orientierung von SH3- und SH2-Domäne ist. Im Rahmen dieser Arbeit wurde mit Hilfe von Moleküldynamik(MD)-Simulationen untersucht, ob die Flexibilität des SH3-SH2 Linkers einen globalen Einfluss auf die Struktur und Dynamik von Src-Kinasen hat. Hierzu wurde die starre SH3-SH2 Linkersequenz aus Hck in der Struktur der inaktiven Kinase durch die flexiblere Linkersequenz aus Lck ersetzt. Dies führte in MD-Simulationen zu einer erhöhten Flexibilität im Vergleich zur Wildtyp-Hck und somit zu einer verringerten Fähigkeit des regulatorischen Domänenpaars die Kinasedomäne in der inaktiven Konformation zu fixieren. Des Weiteren führte der Austausch des SH3-SH2 Domänenlinkers zu konformationellen Änderungen im Aktivierungssegment der Kinasedomäne. Die hierbei eingenommenen alternativen Konformationen zeigten Ähnlichkeiten zur Konformation der Tyrosinkinase Abl, die nach Bindung des Inhibitors Glivec eingenommen wird. Die Sequenz des SH3-SH2 Linkers könnte demzufolge nicht nur die Modulation der Kinaseaktivität, sondern auch die Ligandenbindungseigenschaften im aktiven Zentrum von Src-Kinasen beeinflussen. Dies könnte erklären, warum Lck im Gegensatz zu den anderen Src-Kinasen durch Glivec gehemmt werden kann. Die Aktivierung der Src-Kinasen kann unter anderem durch die Bindung von Liganden an die regulatorischen Domänen vermittelt werden. Zur Charakterisierung der Determinanten der Affinität und Spezifität der molekularen Erkennung wurden MD-Simulationen und darauf aufbauende energetische Analysen durchgeführt. Als Modellsystem wurde hier die Wechselwirkung zwischen dem Tip-Protein aus Herpesvirus saimiri und der SH3-Domäne der Tyrosinkinase Lyn gewählt. Tip bindet mit dem Sequenzmotiv (175PTPPLPPRPANLG187), welches eine PPII-Helix beinhaltet, an die SH3-Domäne. Mit Hilfe eines in silico Alanin-Scans wurde die Rolle aller Nicht-Proline bei der LynSH3-Bindung analysiert. Die dabei erhaltene Reihenfolge der Wichtigkeit für die Bindung konnte anschließend experimentell mittels Fluoreszenzspektroskopischer Messungen bestätigt werden. Außerdem konnte ein modifizierter Ansatz entwickelt werden, der durch die Berücksichtigung entropischer Beiträge auch für die Deletion von terminalen Aminosäuren zuverlässige Aussagen über Bindungsaffinitäten liefert. Mit dieser Strategie konnte gezeigt werden, dass die Verkürzung des Tip-Peptids um die Reste L186/G187 bei der LynSH3-Bindung zu einer schlechteren Bindung führt, wohingegen bei homologen SH3-Domänen kein Effekt beobachtet wurde. Es konnte weiter gezeigt werden, dass die Wechselwirkungen zwischen L186/G187 in Tip und H41 in LynSH3 maßgeblich zu einer Steigerung der Bindungsaffinität beitragen. Das koordinierte Zusammenspiel zwischen Kinasen und Phosphatasen stellt einen wichtigen regulatorischen Mechanismus in biologischen Systemen dar. Die Proteinphosphatase 1 (PP1), eine der bedeutendsten Serin-/Threonin-Phosphatasen, erreicht ihre enzymatische Aktivität und Substratspezifität durch die Bindung verschiedener Interaktionspartner. Diese Interaktion wird primär über ein fünf Aminosäuren langes Sequenzmotiv vermittelt, dessen Eigenschaften bislang nur unzureichend charakterisiert waren, was die Identifizierung neuer Wechselwirkungspartner erschwerte. In der vorliegenden Arbeit wurde das PP1-Bindemotiv mit Hilfe von in silico (Modelling und MD) und in vitro (GST-Pulldown) Analysen eingehend charakterisiert. Es konnte gezeigt werden, dass die Aminosäure an Position +1 einen Ankerpunkt repräsentiert, da sie ein Netzwerk von Salz- und Wasserstoffbrücken zur PP1&#61543;1 bildet. Des Weiteren konnte gezeigt werden, dass die Positionen +1 und +2 im Bindemotiv positiv geladene bzw. polare Aminosäuren tolerieren. Im Gegensatz dazu ließ die Position +3 eine deutliche Präferenz für Valin erkennen. Die größte Variabilität innerhalb des Bindemotivs konnte an der Position +4 beobachtet werden, wohingegen an Position +5 nur die Seitenketten der unpolaren und aromatischen Aminosäuren Phenylalanin und Tryptophan toleriert wurden. Durch den kombinierten Ansatz und die systematische Analyse jeder einzelnen Position des PP1-Bindemotivs war es somit möglich ein spezifisches Interaktionsmotiv [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] aufzustellen. Die Brauchbarkeit dieses Motivs für genomweite Suchen konnte anschließend durch die Identifizierung und experimentelle Verifizierung neuer PP1-Interaktionspartner bestätigt werden. Die HIV-1 Protease, ein Enzym des HI-Virus, ist essentiell für die Bildung reifer Viruspartikel. Somit stellt die Protease ein äußerst wichtiges Zielmolekül für die Entwicklung von Wirkstoffen gegen die Immunschwächekrankheit AIDS dar. Eines der Hauptprobleme der antiretroviralen Therapie ist die schnelle Entwicklung von Resistenzen gegen die eingesetzten Wirkstoffe, welche häufig auf Mutationen der HIV-1 Protease zurückzuführen sind. Zur Entwicklung effektiver und lang anhaltender Wirkstoffe ist es wichtig die Resistenzmechanismen zu verstehen. In dieser Arbeit wurde die Mutation E35D, welche unter anderem im Zusammenhang mit dem Inhibitor Amprenavir beschrieben wird, untersucht. Um einen Einblick in den Resistenzmechanismus zu erhalten, wurden sowohl für den Wildtyp als auch für die Mutante MD-Simulationen der freien und ligandengebundenen Protease durchgeführt. Im Verlauf der Simulationen wurde für die mutierte HIV-1 Protease eine höhere Flexibilität von Schleifenbereichen sichtbar, die Teil der Ligandenbindungstasche sind. Dies wirkte sich bei der freien Protease auf das Gleichgewicht zwischen geschlossener und halboffener Konformation aus, wohingegen bei der ligandengebundenen Protease intermolekulare Wechselwirkungen gestört wurden. Energetische Analysen zeigten, dass die Mutation zu einer signifikanten Erniedrigung der Bindungsenergien für Liganden führt und somit die beobachtete Resistenzentstehung erklären kann. Weiterführende Analysen zeigten, dass die Aminosäure E35 auch in einem Epitop liegt, das für die Erkennung des Virus durch das Immunsystem wichtig ist. Das Virus kann durch Mutationen im Epitop den Erkennungsprozess erschweren und so zu einer Unterdrückung der Immunantwort führen. Die Ergebnisse der Untersuchungen deuten darauf hin, dass die Mutation sowohl Resistenz gegen Proteaseinhibitoren vermittelt als auch eine "Flucht" vor der Erkennung durch das Immunsystem ermöglicht.Protein interactions play an essential function in virtually all biological processes. To understand the underlying molecular principles it is important to characterize the structure and dynamics of proteins on an atomic level. In this work, different protein interactions were analyzed with various in silico and in vitro methods. The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family members of tyrosine kinase, which are found at the beginning of a many signal pathways, differ in their physiological function despite their large structural similarity. In previous work, it was shown that the flexibility of the SH3-SH2 domain linker is critical for the relative orientation of the SH3 and SH2 domains. In this thesis, the global effect of the flexibility of the SH3-SH2 linker on the structure and dynamics of Src kinases was analyzed using of molecular dynamics (MD) simulations. For this purpose, the rigid SH3-SH2 linker sequence of inactive Hck kinase structure was replaced with the more flexible linker sequence from Lck. In the MD simulations, this lead to a higher flexibility in comparison to the wild-type Hck, which, in turn, reduced the regulatory domain pair's ability to fixate the kinase domain in the inactive conformation. Furthermore, the exchange of the SH3-SH2 domain linker lead to conformational changes in the activation segment of the kinase domain. The resulting alternative conformation showed similarities to the conformation of the tyrosine kinase Abl when bound to the inhibitor Glivec. Thus, the sequence of the SH3-SH2 linker can not only modulate the kinase activity, but can also influence the ligand binding properties of the active site of Src kinases. This could explain why Lck is, in contrast to other Src kinases, inhibited by Glivec. One possibility to activate the Src kinases is the binding of ligands to the regulatory domains. To characterize the affinity and specificity determinants of the molecular recognition, MD simulations and, subsequently, energetic analyses were performed. The interaction between the Tip protein of Herpesvirus saimiri and the SH3 domain of tyrosine kinase Lyn was chosen as a model system. Tip binds to the SH3 domain by using the sequence motif (175PTPPLPPRPANLG187), which contains a PPII-helix. The role of all non-prolines was analyzed with the help of an in silico alanine scan. The resulting order of importance with respect to the binding, could be experimentally verified using fluorescence spectroscopy. In addition, by taking entropic contributions into account, a modified approach which allows reliable statements about the effect of terminal ligand truncations was developed. By using this strategy, it was shown that the truncation of the Tip peptide by the L186/G187 residues leads to a lower binding af_nity for LynSH3. In contrast, when binding the truncated Tip peptide to homologous SH3 domains, no changes in the binding affinities were detected. It could furthermore be shown that the interaction between L186/G187 in Tip and H41 in LynSH3 significantly increases the binding affinity. The coordinated interplay between kinases and phosphatases represents a fundamental regulatory mechanism in biological systems. Protein phosphatase 1 (PP1), a major serine/threonine phosphatase, regulates its enzymatic activity and substrate specificity by acquisition of different binding partners. These interactions are primarily mediated by a five residue sequence motif, the properties of which are thus far not well characterized. Detailed knowledge of its sequence properties would greatly facilitate the identification of new interaction partners. In this work, the PP1 docking motif was thoroughly characterized with a combination of in silico (molecular modeling, MD simulations) and in vitro (GST pull-down) analysis. It was shown that the amino acid at position +1 represents a key anchor point of the motif, as it forms a network of salt bridges and hydrogen bonds to PP1. In addition, it could be shown that positions +1 and +2 of the docking motif prefer positively charged and/or polar residues. In contrast, the position +3 clearly favors the hydrophobic amino acid valine. The largest sequence variability was observed at position +4, whereas position +5 only tolerates the unpolar and aromatic side chains of phenylalanine and tryptophan. By using a combined approach and performing a systematic analysis of each individual position of the PP1&#61543;1 docking motif, the specific interaction motif [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] could be defined. The usefulness of this motif for genome-wide searches was subsequently tested by identifying and experimentally verifying previously unknown PP1 interactors. The HIV-1 protease, an enzyme of the HI virus, is essential for replication and assembly of the virus. This property makes it an important target for the design of antiviral agents for AIDS treatment. A major problem of the current HIV therapies is the rapid development of resistance to antiretroviral drugs by genetic mutation. It is necessary to understand the resistance mechanism in order to develop more effective and longer lasting treatment regimens. In this thesis, the mutation E35D, which has been described in context with the inhibitor amprenavir, was analyzed. To gain insight into the resistance mechanism, molecular dynamics simulations of the wild-type's as well as the mutant's free, and ligand complexed proteases were performed. In the course of the simulations, an increased flexibility of the mutated HIV protease's flaps, which are parts of the binding pocket, could be observed. This affects the conformational equilibrium between the closed and semiopen conformations of the free protease, whereas in the ligand bound protease some intermolecular interactions are disrupted. Energetic analysis showed that the mutation also causes a significant reduction of the calculated ligand binding energies, thus giving a plausible explanation for its ability to develop resistance. Further analysis showed that the amino acid E35 is also located in an epitope which is important for the recognition of the virus by the immune system. Mutations in the epitope allow the virus to hamper the recognition process, thereby suppressing an immune system response. The results of the investigation suggest that the mutation conveys resistance against protease inhibitors and also allows the virus to escape immune system recognition