Molecular Modeling of Signal Peptide Recognition by Eukaryotic Sec Complexes

Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways.

Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.

Proteomic characterization of primary and metastatic prostate cancer reveals reduced proteinase activity in aggressive tumors

Prostate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. In order to improve patient outcomes, it is critical to clinically separate aggressive PCa (AG) from non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with aggressive PCa, it is still challenging to separate AG from NAG. To better understand the functional consequences of PCa progression and the unique features of the AG subtype, we studied the proteomic signatures of primary AG, NAG and metastatic PCa. 39 PCa and 10 benign prostate controls in a discovery cohort and 57 PCa in a validation cohort were analyzed using a data-independent acquisition (DIA) SWATH–MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using a validation cohort; and was also compared with a TCGA mRNA expression dataset and confirmed by immunohistochemistry (IHC) using PCa tissue microarray (TMA). 4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed reduced expressions of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates and the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using an independently-collected tumor cohort, correlated with a TCGA mRNA dataset, and confirmed by immunohistochemical stains of PCa tumor microarray (TMA). Our study is the first large-scale proteomics analysis of PCa tissue using a DIA SWATH-MS platform. It provides not only an interrogative proteomic signature of PCa subtypes, but also indicates the critical roles played by certain proteinases during tumor progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in PCa and for the development of a clinical assay to distinguish aggressive from indolent PCa.

Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Proteomic Characterization of Primary and Metastatic Prostate Cancer Reveals Reduced Proteinase Activity in Aggressive Tumors

BackgroundProstate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. Clinically, it is critical to separate and treat aggressive PCa (AG) from non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with PCa, it is still challenge to separate AG from NAG. To better understand the functional consequence of PCa progression and the unique features of AG from NAG, we studied proteomic signatures of primary AG, NAG and metastatic PCa.Methodes39 PCa and 10 benign prostate controls in discovery cohort and 57 PCa in independent-collected validation cohort were analyzed using data-independent acquisition (DIA) SWATH–MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using validation cohort, as well as by comparison with TCGA mRNA expression and immnunochemistry on PCa tissue microarray (TMA).Results4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed the reduced expression of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis using SWATH data further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates in AG tumors, including the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using independent-collected cohort, by comparison with TCGA mRNA data, and the immunochemical stains of our tumor microarray (TMA).ConclusionsOur study is the first large-scale proteomics analysis of PCa tissue using DIA SWATH-MS platform. It not only provides an interrogative proteomic signature of PCa subtypes, but also indicates critical roles of certain proteinases, especially DPP4, in PCa progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in the PCa progression as well as for the development of a clinical assay to distinguish aggressive from indolent PCa.

Label-Free Assay of Protein Kinase A Activity and Inhibition Using a Peptide-Based Electrochemical Sensor

We propose a simple label-free electrochemical biosensor for monitoring protein kinase activity and inhibition using a peptide-modified electrode. The biosensor employs cys-kemptide (CLRRASLG) as a substrate peptide which was immobilized on the surface of a gold electrode via the self-assembly of the thiol terminals in cysteine (C) residues. The interaction between protein kinase A (PKA) and adenosine 5′-triphosphate (ATP) on the cys-kemptide immobilized electrode can cause the transfer of ATP terminal phosphates to the peptide substrates at serine (S) residues, which alters the surface charge of the electrode, thus enabling monitoring of the PKA activity via measuring the interfacial electron transfer resistance with electrochemical impedance spectroscopy. The proposed sensor showed reliable, sensitive, and selective detection of PKA activity with a wide dynamic range of 0.1–100 U/mL and a detection limit of 56 mU/mL. The sensor also exhibited high selectivity, rendering it possible to screen PKA inhibitors. Moreover, the sensor can be employed to evaluate the activity and inhibition of PKA in real samples.

Quercetin 3,5,7,3′,4′-pentamethyl ether from Kaempferia parviflora directly and effectively activates human SIRT1

Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a crucial regulator that produces multiple physiological benefits, such as the prevention of cancer and age-related diseases. SIRT1 is activated by sirtuin-activating compounds (STACs). Here, we report that quercetin 3,5,7,3′,4′-pentamethyl ether (KPMF-8), a natural STAC from Thai black ginger Kaempferia parviflora, interacts with SIRT1 directly and stimulates SIRT1 activity by enhancing the binding affinity of SIRT1 with Ac-p53 peptide, a native substrate peptide without a fluorogenic moiety. The binding affinity between SIRT1 and Ac-p53 peptide was enhanced 8.2-fold by KPMF-8 but only 1.4-fold by resveratrol. The specific binding sites of KPMF-8 to SIRT1 were mainly localized to the helix2–turn–helix3 motif in the N-terminal domain of SIRT1. Intracellular deacetylase activity in MCF-7 cells was promoted 1.7-fold by KPMF-8 supplemented in the cell medium but only 1.2-fold by resveratrol. This work reveals that KPMF-8 activates SIRT1 more effectively than resveratrol does.

A new Förster resonance energy transfer-based platform to track substrate ubiquitination by fluorescence

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence based techniques in ubiquitination studies remain underexplored at least partly due to challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, Förster resonance energy transfer (FRET) di-ubiquitination, to track substrate ubiquitination by fluorescence.This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The di-ubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET di-ubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method one is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method two is a chemo-enzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based di-ubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.

Factors Underlying Asymmetric Dynamics of Disaggregase and Microtubule Severing AAA+ Machines

Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring–shaped hexamers that enable protein remodeling by coupling large–scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed these motions and intra-ring interactions that support them by performing extensive explicit solvent molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexamers of severing proteins and of the nucleotide binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central PL1 loops of the hexamer. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong inter-protomer collaboration. Severing proteins have weaker inter-protomer coupling and stronger intra-protomer stabilization through salt bridges formed between PL2 and PL3 loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker inter–protomer coupling through salt bridges formed by non–canonical loops and stronger intra–protomer coupling. Pore width fluctuations associated with the PL1 constriction in the spiral states, in the presence of a substrate peptide, highlight stark differences between narrowing of channels of severing proteins and widening of the NBD1 ring of ClpB. This indicates divergent substrate processing mechanisms of remodeling and translocation by ClpB and substrate tail-end gripping and possible wedging on microtubule lattice by severing enzymes. Relaxation dynamics of the distance between the PL1 loops and the centers of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens of microseconds on millisecond experimental timescales. For ClpB the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.

Targeting Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1: A Structure-based Virtual Screening Approach to Find Novel Inhibitors

Background : Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is an enzyme that isomerizes phosphorylated serine or threonine motifs adjacent to proline residues. Pin1 has important roles in several cellular signaling pathways, consequently impacting the development of multiple types of cancers. Methods: Based on the previously reported inhibitory activity of pentacyclic triterpenoids isolated from the gum resin of Boswellia genus against Pin1, we designed a computational experiment using molecular docking, pharmacophore filtering, and structural clustering allied to molecular dynamics (MD) simulations and binding free energy calculations to explore the inhibitory activity of new triterpenoids against Pin1 structure. Results: Here, we report different computational evidence that triterpenoids from neem (Azadirachta indica A. Juss), such as 6-deacetylnimbinene, 6-Oacetylnimbandiol, and nimbolide, replicate the binding mode of the Pin1 substrate peptide, interacting with high affinity with the binding site and thus destabilizing the Pin1 structure. Conclusion: Our results are supported by experimental data, and provide interesting structural insights into their molecular mechanism of action, indicating that their structural scaffolds could be used as a start point to develop new inhibitors against Pin1.