Here we report the use of a self-assembling protein cage to sequester and solubilize recombinant proteins which are usually trafficked to insoluble inclusion bodies. Our results suggest that protein ...cages can be used as novel vehicles to rescue and produce soluble proteins that are otherwise difficult to obtain using conventional methods.
Here we report the use of a self-assembling protein cage to sequester and solubilize recombinant proteins which are usually trafficked to insoluble inclusion bodies. Our results suggest that protein ...cages can be used as novel vehicles to rescue and produce soluble proteins that are otherwise difficult to obtain using conventional methods.
Catalytically active enzymes and proteins are rescued from inclusion body formation and rapidly purified by sequestration inside the P22 VLP.
The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of ...tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.
This dissertation focuses largely on structure-function relationship of microtubules, with a supplemental focus on bacterial nanocompartments known as encapsulins. Microtubules (MTs) are an essential ...component of the eukaryotic cell. MTs are crucial for intracellular trafficking, cellular division, and motility, along with many other functions within the cell. The building blocks of a MT are tubulin heterodimers, which are GTPases that self-assemble to form a hollow, cylindrical MT architecture. The diversity of MT functions is achieved in part by a curious phenomenon known as dynamic instability whereby MTs are in a constant state of flux between growth and catastrophic depolymerization. These dynamics are directly linked to the nucleotide state of the MT, whereby the interplay the GTP and GDP nucleotide states determines the propensity for growth or shrinkage. The intrinsic regulation of dynamic instability, in addition to extrinsic regulation by various MT-associated proteins (MAPs), is absolutely critical for proper cellular function. Cryo-electron microscopy (cryo-EM) was used to directly visualize these important biological assemblies in their native states. The first aspect of my work was to determine how the nucleotide state or the binding of common MAPs (EBs and Kinesins), affected the MT structure. Previous MT structures required MAP-binding to act as fiducials in order to facilitate high-resolution, and Chapter One of this dissertation shows the first high-resolution structures of undecorated MTs bound to various different nucleotides. The next aspect of my work was to examine the GTP-bound MT structure, in order to learn how certain MAPs specifically recognize the GTP state, and the conformational dynamics that occur upon GTP hydrolysis. Prior to this study, the MT field has used a GTP analog, GMPCPP, as a proxy for the GTP bound state. However, no one had actually observed MTs in the GTP state. Chapter Two in this dissertation uses mutated recombinant human tubulin that is hydrolysis-deficient to trap MTs that are truly GTP-bound. I found that these tubulin mutants are an invaluable platform for studying MTs. However, it appears that some mutants appear to create non-physiological assemblies, creating caveats for this system similar to the imperfections of nucleotide analogs. The last chapter of this dissertation is the culmination of work using cryo-EM to better understand the assembly principles for a bacterial nanocompartment known as the encapsulin. Encapsulins were first discovered in 2008, and are typically composed of an enzymatic cargo confined within a proteinaceous shell. This compartmentalization can provide the cell with protection from toxic intermediates, or help with increasing local concentration to make the reaction more efficient. The methods driving cargo encapsulation to support these diverse functions remained unknown. For one encapsulin species, I determined how the cargo enzyme was associated with the encapsulin shell, and found that encapsulin cargos do indeed use a form of symmetry-matching. In this experiment, I found that a pentameric cargo protein binds at the pentameric vertices of the encapsulin shell, thus regulating where the cargo is within the compartment as well as the stoichiometry of cargo encapsulation. Serendipitously, while purifying this encapsulin, I also realized it was a previously overlooked flavoprotein. Given the fact that the cargo enzyme is a ferritin-like protein performing redox chemistry, this flavoprotein designation is very intriguing and warrants additional experiments. Taken together this dissertation utilizes cryo-EM as a tool to probe the structural underpinnings that drive microtubule and encapsulin assembly and function.
Objective
We recently reported successful treatment of a child with febrile infection‐related epilepsy syndrome (FIRES), a subtype of new onset refractory status epilepticus, with the recombinant ...interleukin‐1 (IL1) receptor antagonist (IL1RA) anakinra. On this basis, we tested whether endogenous IL1RA production or function is deficient in FIRES patients.
Methods
Levels of IL1β and IL1RA were measured in serum and cerebrospinal fluid (CSF). The inhibitory activity of endogenous IL1RA was assessed using a cell‐based reporter assay. IL1RN gene variants were identified by sequencing. Expression levels for the secreted and intracellular isoforms of IL1RA were measured in patient and control cells by real‐time polymerase chain reaction.
Results
Levels of endogenous IL1RA and IL1β were elevated in the serum and CSF of patients with FIRES (n = 7) relative to healthy controls (n = 10). Serum from FIRES patients drove IL1R signaling activity and potentiated IL1R signaling in response to exogenous IL1β in a cell‐based reporter assay. Functional assessment of endogenous IL1RA activity in 3 FIRES patients revealed attenuated inhibition of IL1R signaling. Sequencing of IL1RN in our index patient revealed multiple variants. This was accompanied by reduced expression of intracellular but not secreted isoforms of IL1RA in the patient's peripheral blood mononuclear cells.
Interpretation
Our findings suggest that FIRES is associated with reduced expression of intracellular IL1RA isoforms and a functional deficiency in IL1RA inhibitory activity. These observations may provide insight into disease pathogenesis for FIRES and other inflammatory seizure disorders and may provide a valuable biomarker for therapeutic decision‐making. Ann Neurol 2019;85:526–537
Some individuals with functional neurological disorder (FND) exhibit motor and affective disturbances, along with limbic hyper-reactivity and enhanced motor-limbic connectivity. Given that the ...multimodal integration network (insula, dorsal cingulate, temporoparietal junction (TPJ)) is implicated in convergent sensorimotor, affective and interoceptive processing, we hypothesised that patients with FND would exhibit altered motor and amygdalar resting-state propagation to this network. Patient-reported symptom severity and clinical outcome were also hypothesised to map onto multimodal integration areas.
Between-group differences in primary motor and amygdalar nuclei (laterobasal, centromedial) were examined using graph-theory stepwise functional connectivity (SFC) in 30 patients with motor FND compared with 30 healthy controls. Within-group analyses correlated functional propagation profiles with symptom severity and prospectively collected 6-month outcomes as measured by the Screening for Somatoform Symptoms Conversion Disorder subscale and Patient Health Questionnaire-15 composite score. Findings were clusterwise corrected for multiple comparisons.
Compared with controls, patients with FND exhibited increased SFC from motor regions to the bilateral posterior insula, TPJ, middle cingulate cortex and putamen. From the right laterobasal amygdala, the FND cohort showed enhanced connectivity to the left anterior insula, periaqueductal grey and hypothalamus among other areas. In within-group analyses, symptom severity correlated with enhanced SFC from the left anterior insula to the right anterior insula and TPJ; increased SFC from the left centromedial amygdala to the right anterior insula correlated with clinical improvement. Within-group associations held controlling for depression, anxiety and antidepressant use.
These neuroimaging findings suggest potential candidate neurocircuit pathways in the pathophysiology of FND.
The pathogenic contribution of neuroinflammation to ictogenesis and epilepsy may provide a therapeutic target for reduction of seizure burden in patients that are currently underserved by traditional ...anti-seizure medications. The Theiler's murine encephalomyelitis virus (TMEV) model has provided important insights into the role of inflammation in ictogenesis, but questions remain regarding the relative contribution of microglia and inflammatory monocytes in this model.
Female C57BL/6 mice were inoculated by intracranial injection of 2 × 10
, 5 × 10
, 1.25 × 10
, or 3.125 × 10
plaque-forming units (PFU) of the Daniel's strain of TMEV at 4-6 weeks of age. Infiltration of inflammatory monocytes, microglial activation, and cytokine production were measured at 24 h post-infection (hpi). Viral load, hippocampal injury, cognitive performance, and seizure burden were assessed at several timepoints.
The intensity of inflammatory infiltration and the extent of hippocampal injury induced during TMEV encephalitis scaled with the amount of infectious virus in the initial inoculum. Cognitive performance was preserved in mice inoculated with 1.25 × 10
PFU TMEV relative to 2 × 10
PFU TMEV, but peak viral load at 72 hpi was equivalent between the inocula. CCL2 production in the brain was attenuated by 90% and TNFα and IL6 production was absent in mice inoculated with 1.25 × 10
PFU TMEV. Acute infiltration of inflammatory monocytes was attenuated by more than 80% in mice inoculated with 1.25 × 10
PFU TMEV relative to 2 × 10
PFU TMEV but microglial activation was equivalent between groups. Seizure burden was attenuated and the threshold to kainic acid-induced seizures was higher in mice inoculated with 1.25 × 10
PFU TMEV but low-level behavioral seizures persisted and the EEG exhibited reduced but detectable abnormalities.
The size of the inflammatory monocyte response induced by TMEV scales with the amount of infectious virus in the initial inoculum, despite the development of equivalent peak infectious viral load. In contrast, the microglial response does not scale with the inoculum, as microglial hyper-ramification and increased Iba-1 expression were evident in mice inoculated with either 1.25 × 10
or 2 × 10
PFU TMEV. Inoculation conditions that drive inflammatory monocyte infiltration resulted in robust behavioral seizures and EEG abnormalities, but the low inoculum condition, associated with only microglial activation, drove a more subtle seizure and EEG phenotype.
Affective symptoms influence health status (health-related quality of life) in functional neurological disorder (FND), and the salience network is implicated in the pathophysiology of FND and ...mood/anxiety disorders. We hypothesised that self-reported health status and affective symptoms would map onto salience network regions and that patients with FND would show decreased insular volumes compared with controls.
This voxel-based morphometry study investigated volumetric differences in 26 patients with FND (21 women, 5 men; mean age=40.3±11.5) compared with 27 healthy controls (22 women, 5 men; mean age=40.5±10.8). Post hoc analyses stratified patients with FND by mental and physical health scores (Short Form Health Survey-36). Within-group analyses investigated associations with mental health, physical health, trait anxiety and depression in patients with FND.
There were no volumetric differences between the complete FND cohort and controls. In stratified analyses, however, patients with FND reporting the most severe physical health impairments showed reduced left anterior insular volume compared with controls. In within-group analyses, impaired mental health and elevated trait anxiety were associated with increased right amygdalar volumes in patients with FND. The relationship between amygdalar volume and mental health, driven by emotional well-being deficits and role limitations due to emotional problems, was independent of sensorimotor functional neurological symptom severity and motor FND subtype. In secondary within-group analyses, increased periaqueductal grey volume was associated with role limitations due to emotional problems. Impaired physical functioning correlated with decreased left anterior insular volumes.
These findings support roles for several regions of the salience network in the pathophysiology of FND.
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