Recruitment of the TolA protein to cell constriction sites in Escherichia coli via three separate mechanisms, and a critical role for FtsWI activity in recruitment of both TolA and TolQ.

The Tol-Pal system of Gram-negative bacteria helps maintain integrity of the cell envelope and ensures that invagination of the envelope layers during cell fission occurs in a well-coordinated manner. In E. coli , the five Tol-Pal proteins (TolQ, R, A, B and Pal) accumulate at cell constriction sites in a manner that normally requires the activity of the cell constriction initiation protein FtsN. While septal recruitment of TolR, TolB and Pal also requires the presence of TolQ and/or TolA, each of the the latter two can recognize constriction sites independently of the other system proteins. What attracts TolQ or TolA to these sites is unclear. We show that FtsN attracts both proteins in an indirect fashion, and that PBP1A, PBP1B and CpoB are dispensable for their septal recruitment. However, the β-lactam aztreonam readily interferes with septal accumulation of both TolQ and TolA, indicating that FtsN-stimulated production of septal peptidoglycan by the FtsWI synthase is critical to their recruitment. We also discovered that each of TolA's three domains can recognize division sites in a separate fashion. Notably, the middle domain (TolAII) is responsible for directing TolA to constriction sites in the absence of other Tol-Pal proteins and CpoB, while recruitment of TolAI and TolAIII requires TolQ and a combination of TolB, Pal, and CpoB, respectively. Additionally, we describe the construction and use of functional fluorescent sandwich fusions of the ZipA division protein, which should be more broadly valuable in future studies of the E. coli cell division machinery. IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction will eventually occur. In the well-studied bacterium Escherichia coli , this machinery contains over thirty distinct proteins. We studied how two such proteins, TolA and TolQ, which also play a role in maintaining integrity of the outer-membrane, are recruited to the machinery. We find that TolA can be recruited by three separate mechanisms, and that both proteins rely on the activity of a well-studied cell division enzyme for their recruitment.

Cryo-EM structure of human Nup155 reveals the biochemical basis for atrial fibrillation linked genetic mutation R391H

SummaryHuman nuclear pore complexes are composed of ∼32 distinct nucleoporins to facilitate bidirectional nucleo-cytoplasmic transport. Many of them have been associated with various human diseases such as an inherited mutation (R391H) in Nup155 is shown as the clinical cause of atrial fibrillation and sudden cardiac arrest. Due to the lack of structural knowledge and mechanistic insights, the roles of Nups in NPC assembly and relevance in human diseases are very restricted. Here, we show the cryo-EM structure of human Nup155 at 5.2-5.7. Å resolution deciphered from 3 distinct particle classes: N-terminus (19-863), C-terminus (864-1337), and longer N-terminus (19-1069). It revealed intrinsic plasticity at the middle domain of Nup155 and the role of species-specific loop regions in an atypical 7-bladed β-propeller domain to provide a distinct interface for Nup93 and Nup35. Due to the proximity of these Nups interacting sites near the Arginine-391 position, atrial fibrillation linked genetic mutation (R391H) causes dissociation from NPC in absence of N-terminal 112 residues.HighlightsCryo-EM structure of human Nup155 at 5.2 Å resolutionSeven bladed β-propeller domain at N-terminus of Nup155 exhibited distinct features for interaction with Nup35 and Nup93The middle domain of Nup155 is highly dynamic in natureStructural mapping allows mechanistic interpretation of AF linked R391H mutation

Numerical Investigation for The Valorization of Waste from HMD Cell Using Electrodialysis

The Hydromagnetic desalination (HMD) system is a continuous process with several advantages, including a high water recovery ratio, and can be favored economically by producing several industrial byproducts instead of discharging the highly concentrated brine to the environment. In the current work, the ions concentration in the Electrodialysis (ED) technique is simulated using COMSOL Multiphysics V.5.2 software. The ED cell simulated in this paper contains two selective membranes (anion and cation) with a width of 0.25 mm each. The salt is to be taken away in the middle domain. The cell operation has been simulated to separate the sodium and chlorine ions from the HMD brine waste solution at 40 or 55ºC temperatures at different voltages and concentrations. In this two-dimensional model, the Nernst-Plank equation has been used to describe ion flux and charge transport in the electrolyte solution. Secondary current distribution theory and the electroneutrality condition have been used in the mathematical model. Finally, Donnan equations have been used to provide the exact fulfillment of boundary conditions for constant voltage mode. The simulation shows that the highest efficiency is obtained at high temperatures and voltage with the lowest feed concentration. Finally, the results have been validated using experimental data from the literature, and a satisfying agreement has been found.

Structural probing of Hsp26 activation and client binding by quantitative cross-linking mass spectrometry

Small heat-shock proteins (sHSP) are important members of the cellular stress response in all species. Their best described function is the binding of early unfolding states and the resulting prevention of protein aggregation. Most sHSPs exist as oligomers but vary in size and subunit organization. Many sHSPs exist as a polydisperse composition of oligomers which undergoes changes in subunit composition, folding status and relative distribution upon heat activation. To date only an incomplete picture of the mechanism of sHSP activation exists and in particularly the molecular basis of how sHSPs bind client proteins and mediate client specificity is not fully understood. In this study we have applied cross-linking mass spectrometry (XL-MS) to obtain detailed structural information on sHSP activation and client binding for yeast Hsp26. Our cross-linking data reveals the middle domain of Hsp26 as client-independent interface in multiple Hsp26::client complexes and indicates that client-specificity is likely mediated via additional binding sites within its αCD and CTE. Our quantitative XL-MS data underpins the middle domain as the main driver of heat-induced activation and client binding but shows that global rearrangements spanning all domains of Hsp26 are taking place simultaneously. We also investigated a Hsp26::client complex in the presence of Ssa1 (Hsp70) and Ydj1(Hsp40) at the initial stage of refolding and observe that the interaction between refolding chaperones is altered by the presence of a client protein, pointing to a mechanism where interaction of Ydj1 with the HSP::client complex initiates assembly of the active refolding machinery.

Aggregation and Its Influence on the Bioactivities of a Novel Antimicrobial Peptide, Temporin-PF, and Its Analogues

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

SummaryThe Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.