Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject ...substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.
LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are ...key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the 'x-Φ-G-Φ' motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.
Abstract
GABA
A
receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms ...underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for β3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the β3 subunit’s extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by β3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABA
A
receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABA
A
receptor gating.
Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient phototransduction and vision. Rhodopsin, the visual pigment in rod photoreceptors, is densely ...packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane, at the surface, in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors.
Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical ...for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.
There is an ongoing need for field-deployable biosensor devices. We have constructed a fully self-contained, hand-held biosensor, based on the surface plasmon resonance technique. The dimensions of ...the sensor unit are 15
×
8
cm, the weight is 600
g and it is powered by a 9
V battery. We have characterised the responsiveness of the sensor using calibrated sucrose solutions and were able to measure changes as small as 3.3
×
10
−6 refractive index units. To demonstrate functionality of the sensor, we have prepared surfaces with an antibody fragment specific for the biological toxin ricin. We were able to detect ricin at 200
ng/mL in 10
min, which is approximately 2500 times less than the minimum lethal dose. We were also able to verify positive binding within a second 10
min window. This sensor demonstrates important steps required for the development of fully integrated, hand-held sensor devices and will form the basis of a multi-analyte system, to be developed in the near future. It also represents the first completely hand-held SPR device, not requiring external power or a computer connection to operate.
Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs ...by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.
The metabolism of archaeal methanogens drives methane release into the environment and is critical to understanding global carbon cycling. Methanogenesis operates at a very low reducing potential ...compared to other forms of respiration and is therefore critical to many anaerobic environments. Harnessing or altering methanogen metabolism has the potential to mitigate global warming and even be utilized for energy applications.
Here, we report draft genome sequences for the isolated methanogens Methanobacterium bryantii, Methanosarcina spelaei, Methanosphaera cuniculi, and Methanocorpusculum parvum. These anaerobic, methane-producing archaea represent a diverse set of isolates, capable of methylotrophic, acetoclastic, and hydrogenotrophic methanogenesis. Assembly and analysis of the genomes allowed for simple and rapid reconstruction of metabolism in the four methanogens. Comparison of the distribution of Clusters of Orthologous Groups (COG) proteins to a sample of genomes from the RefSeq database revealed a trend towards energy conservation in genome composition of all methanogens sequenced. Further analysis of the predicted membrane proteins and transporters distinguished differing energy conservation methods utilized during methanogenesis, such as chemiosmotic coupling in Msar. spelaei and electron bifurcation linked to chemiosmotic coupling in Mbac. bryantii and Msph. cuniculi.
Methanogens occupy a unique ecological niche, acting as the terminal electron acceptors in anaerobic environments, and their genomes display a significant shift towards energy conservation. The genome-enabled reconstructed metabolisms reported here have significance to diverse anaerobic communities and have led to proposed substrate utilization not previously reported in isolation, such as formate and methanol metabolism in Mbac. bryantii and CO
metabolism in Msph. cuniculi. The newly proposed substrates establish an important foundation with which to decipher how methanogens behave in native communities, as CO
and formate are common electron carriers in microbial communities.
Background Posterior ankle impingement syndrome (PAIS) was first described in ballet dancers but is increasingly being diagnosed in
other sports. Operative treatment may be indicated when ...nonoperative measures have failed. Traditionally, operative treatment
has involved an open approach; more recently, posterior ankle arthroscopy has been employed.
Purpose This study was conducted to describe the factors that influence return to play in professional athletes after posterior ankle
arthroscopy for posterior ankle impingement syndrome.
Study Design Case series; Level of evidence 4.
Methods A consecutive series of 28 elite professional soccer players who had clinically and radiologically diagnosed posterior ankle
impingement syndrome that failed to respond to nonoperative treatment underwent posterior ankle arthroscopy for bony or soft
tissue posterior ankle impingement syndrome over 5 years.
Results Of the 28 players, 27 were available for follow-up. Five had a diagnosis of soft tissue impingement and underwent debridement
with flexor hallucis longus release, 13 had a symptomatic os trigonum that was excised arthroscopically, and 9 had removal
of a bony avulsion fragment from the posterior ankle ligament complex. The mean length of time to return to training postoperatively
was 34 days and return to playing was 41 days (range, 29â72 days). The duration of symptoms before surgery and excision of
bony impingement were significantly correlated with the time to return to training and playing. There were no major complications
and no reoperations at an average of 23 months of follow-up (range, 15â49 months).
Conclusion Posterior ankle arthroscopy is safe and effective in the treatment of posterior ankle impingement syndrome in the elite soccer
player, with return to training expected at an average of 5 weeks.
Management of infections in the immunocompromised patient requires unique considerations that are not typically seen in the immunocompetent. Immunocompromised hosts require a broad set of ...differential diagnoses when presenting with febrile illness involving a wide variety of microbiology. Moreover, fungal infections are common, and cotreatment of fungal and bacterial infections occurs with regularity. Fungal coinfection, however, is rare. Here, we describe a patient with Aspergillus and recurrent Cryptococcus neoformans coinfection following completion of treatment for pulmonary cryptococcosis.
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