Relaxation time asymmetry in stator dynamics of the bacterial flagellar motor

The bacterial flagellar motor (BFM) is the membrane-embedded rotary molecular motor which turns the flagellum that provides thrust to many bacterial species. This large multimeric complex, composed of a few dozen constituent proteins, has emerged as a hallmark of dynamic subunit exchange. The stator units are inner-membrane ion channels which dynamically bind and unbind to the peptidoglycan at the rotor periphery, consuming the ion motive force (IMF) and applying torque to the rotor when bound. The dynamic exchange is known to be a function of the viscous load on the flagellum, allowing the bacterium to dynamically adapt to its local viscous environment, but the molecular mechanisms of exchange and mechanosensitivity remain to be revealed. Here, by actively perturbing the steady-state stator stoichiometry of individual motors, we reveal a stoichiometry-dependent asymmetry in stator remodeling kinetics. We interrogate the potential effect of next-neighbor interactions and local stator unit depletion and find that neither can explain the observed asymmetry. We then simulate and fit two mechanistically diverse models which recapitulate the asymmetry, finding stator assembly dynamics to be particularly well described by a two-state catch-bond mechanism.

Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

Mitochondrial ClpP is a serine protease located in the mitochondrial matrix. This protease participates in mitochondrial protein quality control by degrading misfolded or damaged proteins, thus maintaining normal metabolic function. Mitochondrial ClpP is a stable heptamer ring with peptidase activity that forms a multimeric complex with the ATP-dependent unfoldase ClpX (ClpXP) leading to proteolytic activity. Emerging evidence demonstrates that ClpXP is over-expressed in hematologic malignancies and solid tumors and is necessary for the viability of a subset of tumors. In addition, both inhibition and hyperactivation of ClpXP leads to impaired respiratory chain activity and causes cell death in cancer cells. Therefore, targeting mitochondrial ClpXP could be a novel therapeutic strategy for the treatment of malignancy. Here, we review the structure and function of mitochondrial ClpXP as well as strategies to target this enzyme complex as a novel therapeutic approach for malignancy.

Translation reprogramming by eIF3 linked to glioblastoma resistance

Intrinsic resistance to current therapies, leading to dismal clinical outcomes, is a hallmark of glioblastoma multiforme (GBM), the most common and aggressive brain tumor. Understanding the underlying mechanisms of such malignancy is, therefore, an urgent medical need. Deregulation of the protein translation machinery has been shown to contribute to cancer initiation and progression, in part by driving selective translational control of specific mRNA transcripts involved in distinct cancer cell behaviors. Here, we focus on eIF3, a multimeric complex with a known role in the initiation of translation and that is frequently deregulated in cancer. Our results show that the deregulated expression of eIF3e, the e subunit of eIF3, in specific GBM regions could impinge on selective protein synthesis impacting the GBM outcome. In particular, eIF3e restricts the expression of proteins involved in the response to cellular stress and increases the expression of key functional regulators of cell stemness. Such a translation program can therefore serve as a double-edged sword promoting GBM tumor growth and resistance to radiation.

Galectin-3 in Inflammasome Activation and Primary Biliary Cholangitis Development

Primary biliary cholangitis (PBC) is a chronic inflammatory autoimmune liver disease characterized by inflammation and damage of small bile ducts. The NLRP3 inflammasome is a multimeric complex of proteins that after activation with various stimuli initiates an inflammatory process. Increasing data obtained from animal studies implicate the role of NLRP3 inflammasome in the pathogenesis of various diseases. Galectin-3 is a β-galactoside-binding lectin that plays important roles in various biological processes including cell proliferation, differentiation, transformation and apoptosis, pre-mRNA splicing, inflammation, fibrosis and host defense. The multilineage immune response at various stages of PBC development includes the involvement of Gal-3 in the pathogenesis of this disease. The role of Galectin-3 in the specific binding to NLRP3, and inflammasome activation in models of primary biliary cholangitis has been recently described. This review provides a brief pathogenesis of PBC and discusses the current knowledge about the role of Gal-3 in NLRP3 activation and PBC development.

Determining the correct stoichiometry of Kv2.1/Kv6.4 heterotetramers, functional in multiple stoichiometrical configurations

The electrically silent (KvS) members of the voltage-gated potassium (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9 selectively modulate Kv2 subunits by forming heterotetrameric Kv2/KvS channels. Based on the reported 3:1 stoichiometry of Kv2.1/Kv9.3 channels, we tested the hypothesis that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. We investigate the Kv2.1/Kv6.4 stoichiometry using single subunit counting and functional characterization of tetrameric concatemers. For selecting the most probable stoichiometry, we introduce a model-selection method that is applicable for any multimeric complex by investigating the stoichiometry of Kv2.1/Kv6.4 channels. Weighted likelihood calculations bring rigor to a powerful technique. Using the weighted-likelihood model-selection method and analysis of electrophysiological data, we show that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. Within this stoichiometry, the Kv6.4 subunits have to be positioned alternating with Kv2.1 to express functional channels. The variability in Kv2/KvS assembly increases the diversity of heterotetrameric configurations and extends the regulatory possibilities of KvS by allowing the presence of more than one silent subunit.

Two new coral fluorescent proteins of distinct colors for sharp visualization of cell-cycle progression

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided brighter nuclear labeling for monitoring cell cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3’-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for host immune evasion. Our results provide the first direct mechanistic link between base J and regulation of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating polycistronic transcription in trypanosomatids.Author SummaryTrypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription and novel epigenetic marks such as base J and H3.V. The studies also provide the first direct mechanistic link between J modification of DNA at termination sites and regulated Pol II termination and gene expression in kinetoplastids.

Intracellular Trafficking and Translocation of Pertussis Toxin

Pertussis toxin (PT) is a multimeric complex of six proteins. The PTS1 subunit is an ADP-ribosyltransferase that inactivates the alpha subunit of heterotrimeric Gi/o proteins. The remaining PT subunits form a pentamer that positions PTS1 in and above the central cavity of the triangular structure. Adhesion of this pentamer to glycoprotein or glycolipid conjugates on the surface of a target cell leads to endocytosis of the PT holotoxin. Vesicle carriers then deliver the holotoxin to the endoplasmic reticulum (ER) where PTS1 dissociates from the rest of the toxin, unfolds, and exploits the ER-associated degradation pathway for export to the cytosol. Refolding of the cytosolic toxin allows it to regain an active conformation for the disruption of cAMP-dependent signaling events. This review will consider the intracellular trafficking of PT and the order-disorder-order transitions of PTS1 that are essential for its cellular activity.

Substrate complex competition is a regulatory motif that allows NFκB RelA to license but not amplify NFκB RelB

Signaling pathways often share molecular components, tying the activity of one pathway to the functioning of another. In the NFκB signaling system, distinct kinases mediate inflammatory and developmental signaling via RelA and RelB, respectively. Although the substrates of the developmental, so-called noncanonical, pathway are induced by inflammatory/canonical signaling, crosstalk is limited. Through dynamical systems modeling, we identified the underlying regulatory mechanism. We found that as the substrate of the noncanonical kinase NIK, the nfkb2 gene product p100, transitions from a monomer to a multimeric complex, it may compete with and inhibit p100 processing to the active p52. Although multimeric complexes of p100 (IκBδ) are known to inhibit preexisting RelA:p50 through sequestration, here we report that p100 complexes can inhibit the enzymatic formation of RelB:p52. We show that the dose–response systems properties of this complex substrate competition motif are poorly accounted for by standard Michaelis–Menten kinetics, but require more detailed mass action formulations. In sum, although tonic inflammatory signaling is required for adequate expression of the noncanonical pathway precursors, the substrate complex competition motif identified here can prevent amplification of the active RelB:p52 dimer in elevated inflammatory conditions to ensure reliable RelB-dependent developmental signaling independent of inflammatory context.