Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

Recent Advances of Efficient Design of Terahertz Quantum-Cascade Lasers

Terahertz (THz) quantum cascade lasers (QCLs) are electrically pumped and heterostructure based semiconductor laser sources with intersubband transitions of electrons in different layers of the quantum wells and barriers. The THz QCLs have high output power in THz region which make them important from application point of view. Recently intensive research has been carried out by researchers for obtaining efficient designs of THz sources. Most of the researchers have investigated the THz frequency range between 0.1 and 3 THz; however, the output power of the THz sources in the frequency range 3–5 THz is small because of transit time and resistance-capacitance effects. Nevertheless, the present review is focused for the development of efficient THz QCL sources in the frequency range from 3 to 5 THz where one of the major problem of thermal backfilling of the carriers has to be overcome by engineering the heterostructure.

     

The hinge region of the scaffolding protein of cell contacts, zonula occludens protein 1, regulates interacting with various signaling proteins

Zonula occludens protein 1 (ZO-1) is a ubiquitous scaffolding protein, but it is unknown why it functions in very different cellular contacts. We hypothesized that a specific segment, the unique hinge region, can be bound by very different regulatory proteins. Using surface plasmon resonance spectroscopy and binding assays to peptide libraries, we show, for the first time, that the hinge region directly interacts with disparate signal elements such as G-proteins alpha 12 and alpha i2, the regulator of G-protein signaling 5, multifunctional signaling protein ahnak1, and L-type Ca2+-channel beta-2-subunit. The novel binding proteins specifically bound to a coiled coil-helix predicted in the hinge region of ZO-. The interactions were modulated by phosphorylation in the hinge helix. Activation of the G-proteins influenced their association to ZO-1. In colon cells, G alpha i2 and ZO-1 were associated, as shown by coimmunoprecipitation. After cotransfection in kidney cells, G alpha i2 barely colocalized with ZO-1; the colocalization coefficient was significantly increased when epinephrine activated G-protein signaling. In conclusion, proteins with different regulatory potential adhere to and influence cellular functions of ZO-proteins, and the interactions can be modulated via its hinge region and/or the binding proteins.     

Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis

M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.     

Determinants within the turret and pore-loop domains of KCNQ3 K+ channels governing functional activity

KCNQ1-5 (Kv7.1-7.5) subunits assemble to form a variety of functional K⁺ channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by ∼56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA⁺-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba²⁺ block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position “unlocking” the channels into a conductive conformation.     

Klotho Up-regulates Renal Calcium Channel Transient Receptor Potential Vanilloid 5 (TRPV5) by Intra- and Extracellular N-glycosylation-dependent Mechanisms

The anti-aging protein Klotho is a type 1 membrane protein produced predominantly in the distal convoluted tubule. The ectodomain of Klotho is cleaved and secreted into the urine to regulate several ion channels and transporters. Secreted Klotho (sKL) up-regulates the TRPV5 calcium channel from the cell exterior by removing sialic acids from N-glycan of the channel and inhibiting its endocytosis. Because TRPV5 and Klotho coexpress in the distal convoluted tubule, we investigated whether Klotho regulates TRPV5 action from inside the cell. Whole-cell TRPV5-mediated channel activity was recorded in HEK cells coexpressing TRPV5 and sKL or membranous Klotho (mKL). Transfection of sKL, but not mKL, produced detectable Klotho protein in cell culture media. As for sKL, mKL increased TRPV5 current density. The role of sialidase activity of mKL acting inside is supported by findings that mutations of putative sialidase activity sites in sKL and mKL abrogated the regulation of TRPV5 but that the extracellular application of a sialidase inhibitor prevented the regulation of TRPV5 by sKL only. Mechanistically, coexpression with a dominant-negative dynamin II prevented the regulation of TRPV5 by sKL but not by mKL. In contrast, blocking forward trafficking by brefeldin A prevented the effect with mKL but not with sKL. Therefore, Klotho up-regulates TRPV5 from both the inside and outside of cells. The intracellular action of Klotho is likely due to enhanced forward trafficking of channel proteins, whereas the extracellular action is due to inhibition of endocytosis. Both effects involve putative Klotho sialidase activity. These effects of Klotho may play important roles regarding calcium reabsorption in the kidney.