Treponema pallidum (syphilis) antigen TpF1 induces activation of macrophages and accelerates P2X7R-induced NLRP3-dependent release of IL-1β

Background: Syphilis is a chronic infectious disease caused by Treponema pallidum (Tp) infection, which causes local inflammation in the host. TpF1 is an oligomeric protein expressed by the Tp-infected host that can induce the host immune response. There are few studies regarding the role of TpF1 in macrophage activation and the subsequent release of cytokines. Objective: To elucidate the effects of TpF1 on the pathological process of Syphilis. In addition, we explored how purinergic 2X7 (P2X7R) induced NOD-like receptor family protein 3 (NLRP3) -dependent release of interleukin-1β (IL-1β) and the underlying mechanisms. Methods: We explored the influence of TpF1 on cytokine release by macrophages using qRT-PCR and ELISA. The specific phenotype of activated macrophages was determined by flow cytometry. Results: TpF1 was able to activate macrophages and induce the M1 macrophage phenotype. Moreover, TpF1 activated the NLRP3 inflammasome in macrophages, which was mediated by P2X7R. Conclusions: The Tp-induced protein TpF1 is able to induce macrophage activation and P2X7R-induced NLRP3-dependent release of IL-1β. Our findings provide a theoretical basis for clarifying the clinical symptoms and pathogenesis of syphilis.

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a “resting” hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP’s substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

An Up-Scalable and Cost-Effective Methodology for Isolating a Polypeptide Matrix Metalloproteinase-9 Inhibitor from Lupinus albus Seeds

One of the most challenging problems with food-borne bioactive compounds is that there are commonly no cost-effective, generally recognized as safe (GRAS) methods for obtaining gram quantities of their purified forms. Here we aimed at developing a method to isolate deflamin, an oligomeric protein from lupin seeds with anti-inflammatory and anticancer activity through matrix metalloprotease (MMP)-9 inhibition. Our goal was to develop a GRAS method that could be easily up-scalable whilst maintaining deflamin’s activity. A sequential precipitation methodology was developed, using an aqueous extraction, followed by heat denaturation, acid precipitation and solubilization in ethanol. A final precipitation with 90% ethanol yielded a purified protein which was sequenced through mass spectrometry and tested for its MMP inhibitory activity using the Dye-quenched (DQ) gelatin assay and the standard wound healing assay in HT29 cells. The developed method yielded a purified oligomer, which represented 0.1% (w/w) of total dry seed weight and was positively confirmed to be deflamin. It further showed to effectively reduce MMP-9 gelatinolytic activity as well as colon cancer cell migration, hence corroborating the effectiveness of our method. Overall, this is the first reported method for isolating an MMP-9 inhibitor from legume seeds, which is up-scalable to an industrial level, in a cost-effective manner.

Increased Serum AXL Is Associated With Knee Osteoarthritis Severity

Objective: Serum AXL expression and clinical significance in patients with knee osteoarthritis (KOA). Methods: 183 patients with knee osteoarthritis were selected and divided based on the Kellgren-Lawrence (KL) score into KL 0 subgroups (n = 42), KL I-II subgroups (n = 90), and KL III-IV subgroups (n = 51). Healthy volunteers (n= 170) in our hospital were selected as the control group. AXL level in serum was detected by enzyme-linked immunosorbent assay (ELISA). The correlation between serum AXL with severity and clinical indicators of osteoarthritis was analyzed. Results: The level of serum AXL was significantly higher in the osteoarthritis group than that in the control group (P<0.05). In the osteoarthritis patients, serum AXL level was increased with the increase of KL score. Serum AXL level was positively correlated with age, body mass index (BMI), erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), cartilage oligomeric protein (COMP), matrix metalloproteinase-13 (MMP-13), and transforming growth factor-β1 (TGF-β1) levels. The cut-off value for serum AXL was determined as 33.375 ng/mL by receiver operating curve (ROC) analysis. Conclusion: The level of serum AXL in patients with osteoarthritis is abnormally high, which is closely related to the severity of osteoarthritis.

Nanotechnological Applications Based on Bacterial Encapsulins

Encapsulins are proteinaceous nanocontainers, constructed by a single species of shell protein that self-assemble into 20–40 nm icosahedral particles. Encapsulins are structurally similar to the capsids of viruses of the HK97-like lineage, to which they are evolutionarily related. Nearly all these nanocontainers encase a single oligomeric protein that defines the physiological role of the complex, although a few encapsulate several activities within a single particle. Encapsulins are abundant in bacteria and archaea, in which they participate in regulation of oxidative stress, detoxification, and homeostasis of key chemical elements. These nanocontainers are physically robust, contain numerous pores that permit metabolite flux through the shell, and are very tolerant of genetic manipulation. There are natural mechanisms for efficient functionalization of the outer and inner shell surfaces, and for the in vivo and in vitro internalization of heterologous proteins. These characteristics render encapsulin an excellent platform for the development of biotechnological applications. Here we provide an overview of current knowledge of encapsulin systems, summarize the remarkable toolbox developed by researchers in this field, and discuss recent advances in the biomedical and bioengineering applications of encapsulins.

PoseFilter: a PyMOL plugin for filtering and analyzing small molecule docking in symmetric binding sites

Summary ‘PoseFilter’ is a PyMOL plugin that assists in analyses and filtering of docked poses. PoseFilter enables automatic detection of symmetric poses from docking outputs and can be accessed using both graphical user interface and command-line options within the PyMOL program. Two methods of analyses, root mean square deviations and interaction fingerprints, are available from this plugin. The capabilities of the plugin are demonstrated using docking outputs from different oligomeric protein-ligand complexes. Availability and implementation The plugin can be downloaded from the GitHub page, https://github.com/skalyaanamoorthy/PoseFilter. Supplementary information Supplementary data are available at Bioinformatics online.