Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.     

Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.     

Comprehensive association testing of common mitochondrial DNA variation in metabolic disease

Many lines of evidence implicate mitochondria in phenotypic variation: (a) rare mutations in mitochondrial proteins cause metabolic, neurological, and muscular disorders; (b) alterations in oxidative phosphorylation are characteristic of type 2 diabetes, Parkinson disease, Huntington disease, and other diseases; and (c) common missense variants in the mitochondrial genome (mtDNA) have been implicated as having been subject to natural selection for adaptation to cold climates and contributing to "energy deficiency" diseases today. To test the hypothesis that common mtDNA variation influences human physiology and disease, we identified all 144 variants with frequency >1% in Europeans from >900 publicly available European mtDNA sequences and selected 64 tagging single-nucleotide polymorphisms that efficiently capture all common variation (except the hypervariable D-loop). Next, we evaluated the complete set of common mtDNA variants for association with type 2 diabetes in a sample of 3,304 diabetics and 3,304 matched nondiabetic individuals. Association of mtDNA variants with other metabolic traits (body mass index, measures of insulin secretion and action, blood pressure, and cholesterol) was also tested in subsets of this sample. We did not find a significant association of common mtDNA variants with these metabolic phenotypes. Moreover, we failed to identify any physiological effect of alleles that were previously proposed to have been adaptive for energy metabolism in human evolution. More generally, this comprehensive association-testing framework can readily be applied to other diseases for which mitochondrial dysfunction has been implicated.     

SpoIID-Mediated Peptidoglycan Degradation Is Required throughout Engulfment during Bacillus subtilis Sporulation

SpoIID is a membrane-anchored enzyme that degrades peptidoglycan and is essential for engulfment and sporulation in Bacillus subtilis. SpoIID is targeted to the sporulation septum, where it interacts with two other proteins required for engulfment: SpoIIP and SpoIIM. We changed conserved amino acids in SpoIID to alanine to determine whether there was a correlation between the effect of each substitution on the in vivo and in vitro activities of SpoIID. We identified one amino acid substitution, E88A, that eliminated peptidoglycan degradation activity and one, D210A, that reduced it, as well as two substitutions that destabilized the protein in B. subtilis (R106A and K203A). Using these mutants, we show that the peptidoglycan degradation activity of SpoIID is required for the first step of engulfment (septal thinning), as well as throughout membrane migration, and we show that SpoIID levels are substantially above the minimum required for engulfment. The inactive mutant E88A shows increased septal localization compared to the wild type, suggesting that the degradation cycle of the SpoIID/SpoIIP complex is accompanied by the activity-dependent release of SpoIID from the complex and subsequent rebinding. This mutant is also capable of moving SpoIIP across the sporulation septum, suggesting that SpoIID binding, but not peptidoglycan degradation activity, is needed for relocalization of SpoIIP. Finally, the mutant with reduced activity (D210A) causes uneven engulfment and time-lapse microscopy indicates that the fastest-moving membrane arm has greater concentrations of SpoIIP than the slower-moving arm, demonstrating a correlation between SpoIIP protein levels and the rate of membrane migration.Endospore formation is an evolutionarily conserved process that allows Bacillus subtilis and related Gram-positive bacteria to adapt to changes in the environment, such as nutrient depletion. Many dramatic morphological changes occur during sporulation, each requiring a multitude of specialized proteins (reviewed in references 13 and 17). First, a sporulation septum is formed near one of the cell poles, forming two separate compartments of unequal sizes and with differing fates (Fig. ​(Fig.1A).1A). The smaller of the two, the forespore, will eventually become the spore, while the larger, the mother cell, will ultimately lyse. Next, the mother cell membranes move up and around the forespore in the poorly understood process of engulfment. Although this process is superficially similar to eukaryotic engulfment, it is complicated by the thick cell wall that surrounds and separates the two compartments. After engulfment, the migrating membranes pinch off from the mother cell membrane, thereby releasing the forespore into the cytoplasm of the mother cell, where it can be enveloped with protective coat proteins and eventually released into the environment as a mature spore. Sporulation provides an ideal, nonessential system for understanding how bacterial cells are capable of undergoing dramatic morphological changes.Open in a separate windowFIG. 1.Engulfment in B. subtilis. (A) (i) Engulfment begins with formation of an asymmetric septum that divides the cell into the forespore (FS) and mother cell (MC). SpoIID (orange pacman) and SpoIIP (green pacman) peptidoglycan degradation enzymes localize to the center of the septum. (ii) SpoIID and SpoIIP thin the septal peptidoglycan, starting from the center and moving toward the cell edges. SpoIIQ (purple ball) and SpoIIIAH (red ball) form a zipper across the septum, assembling foci behind the leading edges. (iii) The peptidoglycan degradation enzymes localize to the leading edges during membrane migration, while additional SpoIIQ-SpoIIIAH complexes assemble around the forespore. (iv) Engulfment membrane fission occurs at the top of the forespore, releasing the forespore into the mother cell cytoplasm. (B) Burnt-bridge Brownian ratchet model for membrane migration, adapted from earlier studies (1, 7). (C) Schematic representation of the SpoIID domain structure. The transmembrane domain (TM) and putative enzymatic domain, as defined by Pfam (14), are indicated. Amino acid numbers are below the schematic, and mutations causing in vivo phenotypes are indicated by an “X”.Engulfment involves dynamic protein localization and large-scale rearrangements of cellular membranes and peptidoglycan to accommodate internalization of the forespore. The physical basis for engulfment remains unclear, but two separate protein machineries that contribute to engulfment have been discovered. The first module involves the only three proteins known to be required for engulfment under all physiological conditions: SpoIID, SpoIIM, and SpoIIP (16, 24, 35). Zymography assays have demonstrated that both SpoIID and SpoIIP degrade peptidoglycan in vitro (1, 8), and this function is thought to be essential for engulfment in wild-type cells (1, 2, 8). SpoIID and SpoIIP are membrane-spanning proteins that directly interact both in vivo and in vitro, as demonstrated by coimmunoprecipitation and affinity chromatography techniques (2, 8). These studies failed to demonstrate an interaction between SpoIIM and either SpoIID or SpoIIP, perhaps because SpoIIM is an integral membrane protein. However, SpoIIM is required for localization of SpoIID and SpoIIP (2, 8), suggesting that all three proteins interact to form a peptidoglycan degradation module that is essential for engulfment.The second system influencing membrane migration is the SpoIIQ/SpoIIIAH zipper, which is required for engulfment only under certain conditions (2, 7, 38). SpoIIQ is produced in the forespore (23) and SpoIIIAH is produced in the mother cell (19). SpoIIQ and SpoIIIAH interact both in vitro and in vivo via their extracellular domains (6, 7, 10). Because these two proteins are produced in separate compartments, the only possible place for an interaction is the intermembrane space between the mother cell and forespore, forming a protein-protein zipper between the two cells. This zipperlike interaction is necessary for septal localization of SpoIIIAH and other mother cell proteins (6, 10, 18) and is capable of holding the two cells together when peptidoglycan is removed with lysozyme (7). Surprisingly, digestion of the peptidoglycan with lysozyme also allows membrane migration in about half of treated cells, in a process requiring the SpoIIQ/SpoIIIAH zipper but not the SpoIIDMP peptidoglycan degradation module. The SpoIIQ-SpoIIIAH zipper also contributes to engulfment in living cells, since strains lacking SpoIIQ or SpoIIIAH complete engulfment more slowly than the wild type and have synergistic engulfment defects when certain secondary mutations are introduced (2, 7, 38). Together, these results strongly support a role for the SpoIIQ/SpoIIIAH module in engulfment, demonstrating that the zipper contributes to the efficiency of membrane migration even when the SpoIIDMP module is present and functional. They also suggest that the engulfment machinery displays functional redundancy and that the zipper module provides a backup machinery for membrane migration.The precise role of the SpoIIDMP module during engulfment remains unclear. One model proposes that SpoIID and SpoIIP act as a burnt-bridge Brownian ratchet (Fig. ​(Fig.1B)1B) (1, 7). This model asserts that as SpoIID and SpoIIP degrade peptidoglycan, they eliminate their own enzymatic targets, resulting in the absence of substrate in one direction and therefore, overall movement in the opposite direction. As the enzymes move forward toward new targets, the mother cell membranes are dragged along with them because they are anchored in the membrane. This hypothesis predicts that SpoIID and SpoIIP are processive enzymes and that the SpoIIDMP complex could function as a motor, moving along peptidoglycan as a track and pulling the membranes with it (1, 7). A second model predicts that peptidoglycan degradation could simply remove a steric hindrance to membrane migration (such as links between the forespore membrane and the cell wall) and that some other mechanism provides the force required for membrane migration. Although the SpoIIQ-SpoIIIAH module can contribute to membrane migration, these proteins are not always essential for engulfment in intact cells (7, 38), suggesting that another unidentified system must generate the force required for membrane movement if the DMP module does not act as a burnt-bridge Brownian ratchet. Recent evidence suggests that peptidoglycan biosynthesis, which is localized to the leading edge of the engulfing membrane and necessary for membrane migration in the absence of the SpoIIQ-SpoIIIAH proteins, might be this missing force generating mechanism (26).Both models predict that the activities of SpoIID and SpoIIP are essential for membrane migration. This requirement has been demonstrated for SpoIIP (8) and, while this work was under review, for SpoIID. SpoIID shows no sequence similarity to any characterized enzyme that degrades peptidoglycan and thus constitutes the founding member of a new class of enzymes that remodel peptidoglycan (1, 27). However, SpoIID does show some similarity to B. subtilis LytB (24), a protein that enhances the activity of the amidase LytC (5, 20, 34), while SpoIIP is related to LytC (14). A recent study demonstrated that SpoIIP is both an amidase and endopeptidase and that SpoIID both activates SpoIIP and functions as a lytic transglycosylase, cleaving peptidoglycan between NAG and NAM (27). Together, these two enzymes degrade peptidoglycan into its smallest repeating subunits. However, it remains unclear which of the demonstrated or suggested biochemical functions of SpoIID are required for its various in vivo activities (interaction with SpoIIP, localization, septal thinning, and membrane migration), and it is unclear whether peptidoglycan degradation activity is required throughout engulfment or only for the initial stage of septal thinning.We use site-directed mutagenesis to test the role of 56 conserved amino acids in SpoIID, focusing on hydrophilic amino acids that might be involved in protein-protein interactions and peptidoglycan degradation. We identified one mutation (E88A) that eliminates and three others (R106A, K203A, and D210A) that reduce peptidoglycan degradation activity and show that SpoIID activity is required for the earliest stage of engulfment (septal thinning), as well as throughout membrane migration. Our results confirm and extend those of Morlot et al. (27) and also demonstrate that SpoIID activity is required throughout engulfment. Furthermore, our data indicate that the enzymatically inactive mutant protein (E88A) shows increased septal localization compared to the wild-type protein, suggesting that peptidoglycan degradation contributes to the release of SpoIID from the septum. We propose a modified model for the enzymatic cycle of the SpoIID and SpoIIP complex.     

Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel

The TRPV ion channels mediate responses to many sensory stimuli including heat, low pH, neuropeptides, and chemical ligands. All TRPV subfamily members contain an intracellular N-terminal ankyrin repeat domain (ARD), a prevalent protein interaction motif. The 1.6-A crystal structure of the TRPV2-ARD, with six ankyrin repeats, reveals several atypical structural features. Repeats one through three display unusually long and flexible fingers with a large number of exposed aromatic residues, whereas repeats five and six have unusually long outer helices. Furthermore, a large counterclockwise twist observed in the stacking of repeats four and five breaks the regularity of the domain, altering the shape of surfaces available for interactions with proteins or other cellular ligands. Both solution studies and crystal packing interactions indicate that the TRPV2-ARD does not form homo-oligomers, suggesting that the ARD of TRPV ion channels may be used for interactions with regulatory factors rather than in promoting tetrameric assembly of the ion channels.     

Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine?

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-alpha, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS.     

Basic Biology and Mechanisms of Neural Ciliogenesis and the B9 Family

Although the discovery of cilia is one of the earliest in cell biology, the past two decades have witnessed an explosion of new insight into these enigmatic organelles. While long believed to be vestigial, cilia have recently moved into the spotlight as key players in multiple cellular processes, including brain development and homeostasis. This review focuses on the rapidly expanding basic biology of neural cilia, with special emphasis on the newly emerging B9 family of proteins. In particular, recent findings have identified a critical role for the B9 complex in a network of protein interactions that take place at the ciliary transition zone (TZ). We describe the essential role of these protein complexes in signaling cascades that require primary (nonmotile) cilia, including the sonic hedgehog pathway. Loss or dysfunction of ciliary trafficking and TZ function are linked to a number of neurologic diseases, which we propose to classify as neural ciliopathies. When taken together, the studies reviewed herein point to critical roles played by neural cilia, both in normal physiology and in disease.     

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.     

A systems approach to improving rural care in Ethiopia

Background

Multiple interventions have been launched to improve the quality, access, and utilization of primary health care in rural, low-income settings; however, the success of these interventions varies substantially, even within single studies where the measured impact of interventions differs across sites, centers, and regions. Accordingly, we sought to examine the variation in impact of a health systems strengthening intervention and understand factors that might explain the variation in impact across primary health care units.

Methodology/Principal Findings

We conducted a mixed methods positive deviance study of 20 Primary Health Care Units (PHCUs) in rural Ethiopia. Using longitudinal data from the Ethiopia Millennium Rural Initiative (EMRI), we identified PHCUs with consistently higher performance (n = 2), most improved performance (n = 3), or consistently lower performance (n = 2) in the provision of antenatal care, HIV testing in antenatal care, and skilled birth attendance rates. Using data from site visits and in-depth interviews (n = 51), we applied the constant comparative method of qualitative data analysis to identify key themes that distinguished PHCUs with different performance trajectories. Key themes that distinguished PHCUs were 1) managerial problem solving capacity, 2) relationship with the woreda (district) health office, and 3) community engagement. In higher performing PHCUs and those with the greatest improvement after the EMRI intervention, health center and health post staff were more able to solve day-to-day problems, staff had better relationships with the woreda health official, and PHCU communities'' leadership, particularly religious leadership, were strongly engaged with the health improvement effort. Distance from the nearest city, quality of roads and transportation, and cultural norms did not differ substantially among PHCUs.

Conclusions/Significance

Effective health strengthening efforts may require intensive development of managerial problem solving skills, strong relationships with government offices that oversee front-line providers, and committed community leadership to succeed.