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Wnt16 is expressed in mouse & human vascular smooth muscle (VSM). We assessed the impact of Wnt16 deficiency on angiotensin-II (AngII) -induced aneurysmal remodeling. AngII infusion for 4 weeks increased aortic Wnt16 2-fold & ascending thoracic aorta diameter by 60% (p = 1.5E-4, ANOVA p = 3E-6) in male LDLR-/- mice. Wnt16-/-;LDLR-/- cohorts exhibited worsened aorta dilatation (by 20%; Holm-Sidak p =0.03), indicating a protective role for Wnt16. Wnt16 deficiency reduced both pulse wave velocity & systolic blood pressure (Wnt16-null = 132+/- 3 vs. 146 +/- 6 mmHg, p < 0.05, n =11/genotype), suggesting altered contractile function. Since hypomorphic alleles encoding contractile proteins predispose to ascending aortic aneurysms, we studied the impact of Wnt16 on the contractile phenotype. VSM from Wnt16-null thoracic aorta exhibited reductions in Acta2 & SM22 (protein, mRNA; down 50%, p < 0.05), Myh11 (down 60%; p < 0.05), & Myocardin ( Myocd ; by 65%; p =0.0002). Inhibitory H3K27me3 histone methylation was selectively increased 3-fold in chromatin of genes encoding contractile proteins in Wnt16-/- VSM (p = 3E-5). DNA fragmentation was increased 22% in Wnt16-null VSM (p < 0.01). Label-free quantitative proteomic analyses of aorta revealed (a) upregulation of 14 mechanosensitive LIM proteins in Wnt16-null mice, with (b) > 90% reduction in Ankrd1 - a wound contraction protein & a prototypic target of Yap1-Taz/Wwtr1 activity in the Hippo pathway. Wnt16 deficiency significantly increased VSM cytoplasmic Yap1-Taz protein accumulation while reducing nuclear Yap1-Taz. siRNA targeting Taz, but not Yap1, reduced contractile gene expression, phenocopying the actions of Wnt16 siRNA or genetic deficiency. Antagonism of Lats1/2, Hippo pathway kinases that inhibit Yap1-Taz cytoplasmic-nuclear shuttling, upregulated VSM Myocd (4.4x), Acta2 (1.6x), Ankrd1 (2.5x; p < 0.01), mimicking actions of 8 nM Wnt16 treatment. Dosing mice with Wnt16 (0.25 mg/kg/day, 2 days) upregulated aortic contractile gene expression 1.6- to 1.8-fold (all p < 0.05). Wnt16 limits thoracic aortic remodeling & preserves the VSM contractile phenotype in part via the Hippo - Taz/Wwtr1 signal relay. Strategies that augment Wnt16 activity may mitigate enlargement of ascending aortic aneurysms.

Rationale: The PTH1R (PTH [parathyroid hormone]/PTHrP [PTH-related protein] receptor) is expressed in vascular smooth muscle (VSM) and increased VSM PTH1R signaling mitigates diet-induced arteriosclerosis in LDLR −/− mice. Objective: To study the impact of VSM PTH1R deficiency, we generated mice SM22-Cre:PTH1R(fl/fl);LDLR −/− mice (PTH1R-VKO) and Cre-negative controls. Methods and Results: Immunofluorescence and Western blot confirmed PTH1R expression in arterial VSM that was reduced by Cre-mediated knockout. PTH1R-VKO cohorts exhibited increased aortic collagen accumulation in vivo, and VSM cultures from PTH1R-VKO mice elaborated more collagen (2.5-fold; P =0.01) with elevated Col3a1 and Col1a1 expression. To better understand these profibrotic responses, we performed mass spectrometry on nuclear proteins extracted from Cre-negative controls and PTH1R-VKO VSM. PTH1R deficiency reduced Gata6 but upregulated the MADS (MCM1, Agamous, Deficiens, and Srf DNA-binding domain)-box transcriptional co-regulator, Mkl-1 (megakaryoblastic leukemia [translocation] 1). Co-transfection assays ( Col3a1 promoter-luciferase reporter) confirmed PTH1R-mediated inhibition and Mkl-1-mediated activation of Col3a1 transcription. Regulation mapped to a conserved hybrid CT(A/T) 6 GG MADS-box cognate in the Col3a1 promoter. Mutations of C/G in this motif markedly reduced Col3a1 transcriptional regulation by PTH1R and Mkl-1. Upregulation of Col3a1 and Col1a1 in PTH1R-VKO VSM was inhibited by small interfering RNA targeting Mkl1 and by treatment with the Mkl-1 antagonist CCG1423 or the Rock (Rho-associated coiled-coil containing protein kinase)-2 inhibitor KD025. Chromatin precipitation demonstrated that VSM PTH1R deficiency increased Mkl-1 binding to Col3a1 and Col1a1 , but not TNF , promoters. Proteomic studies of plasma extracellular vesicles and VSM from PTH1R-VKO mice identified C1r (complement component 1, r) and C1s (complement component 1, s), complement proteins involved in vascular collagen metabolism, as potential biomarkers. VSM C1r protein and C1r message were increased with PTH1R deficiency, mediated by Mkl-1-dependent transcription and inhibited by CCG1423 or KD025. Conclusions: PTH1R signaling restricts collagen production in the VSM lineage, in part, via Mkl-1 regulatory circuits that control collagen gene transcription. Strategies that maintain homeostatic VSM PTH1R signaling, as reflected in extracellular vesicle biomarkers of VSM PTH1R/Mkl-1 action, may help mitigate arteriosclerosis and vascular fibrosis.

Wnt16 controls bone strength via paracrine osteoblast-osteoclast remodeling interactions. Wnt16 mRNA is highly expressed in both aorta & bone, & immunofluorescence (IF) localized Wnt16 to vascular smooth muscle (VSM). Paracrine VSM - monocyte/macrophage (M/MAC) interactions control arterial remodeling, resembling osteoblast-osteoclast interactions in bone. We assessed the impact of Wnt16 deficiency on aneurysmal remodeling (6-7/group). Angiotensin II infusion for 4 weeks increased ascending thoracic aorta diameter by 60% (Holm-Sidak p = 1.5E-4, ANOVAp = 2.7E-6) in male LDLR-/- mice. Wnt16-/-;LDLR-/- cohorts exhibited worsened ascending aorta dilatation (by 20%; Holm-Sidak p = 0.03), indicating a protective role for Wnt16. Mass spectrometry of thoracic aorta proteins from Wnt16-/-;LDLR-/- vs. LDLR-/-mice revealed a 9-fold increase in MMP12 (p = 0.008; n=3/group) — a M/MAC protease key to aneurysmal remodeling. IF confirmed enhanced adventitial MMP12 in aortas of Wnt16-null mice. Treatment of M/MACs with Wnt16 (8 nM) down-regulated Mmp12 mRNA (78%; p < 0.001), with increased IL10 (2-fold; p < 0.05) & Arg1 (6-fold; p =0.001). Hypomorphic alleles encoding contractile proteins predispose to hereditary thoracic aortic aneurysm; thus, we studied the impact of Wnt16 deficiency on the contractile phenotype. VSM from Wnt16-null thoracic aorta exhibited reductions in Myh11 (down 60%; p < 0.05), Acta2 (by 35%, p Myocardin ( by 65%; p = 0.0002), while Axin2 was increased 1.7-fold (p = 0.01). Acute siRNA-mediated knockdown of Wnt16 in VSM -M/MAC admixtures (95% VSM, 5% M/MAC) also downregulated contractile genes & upregulated Mmp12 . This was phenocopied by siRNA targeting Taz/Wwtr1 — a transcriptional collaborator of Myocd we identified as exhibiting impaired nuclear localization in Wnt16-null VSM. In vivo dosing with Wnt16 (0.25 mg/kg/day, 2 days) upregulated aortic contractile gene expression 1.6- to 1.8-fold, with Mmp12 reduced 50% (p < 0.05), as consistent with the reciprocal changes upon Wnt16 deficiency. Wnt16 limits thoracic aortic remodeling, preserves the VSM contractile phenotype, & reduces Mmp12. Strategies that augment Wnt16 signaling may mitigate enlargement of thoracic aortic aneurysms.

The bacterial mechanosensitive channel of large conductance (MscL) serves as a biological emergency release valve, preventing the occurrence of cell lysis caused by acute osmotic stress. Its tractable nature allows it to serve as a paradigm for how a protein can directly sense membrane tension. Although much is known of the importance of the hydrophobicity of specific residues in channel gating, it has remained unclear whether electrostatics at the membrane plays any role. We studied MscL chimeras derived from functionally distinct orthologues: Escherichia coli and Staphylococcus aureus. Dissection of one set led to an observation that changing the charge of a single residue, K101, of E. coli (Ec)-MscL, effects a channel phenotype: when mutated to a negative residue, the channel is less mechanosensitive and has longer open dwell times. Assuming electrostatic interactions, we determined whether they are due to protein–protein or protein–lipid interactions by performing site-directed mutagenesis elsewhere in the protein and reconstituting channels into defined lipids, with and without negative head groups. We found that although both interactions appear to play some role, the primary determinant of the channel phenotype seems to be protein–lipid electrostatics. The data suggest a model for the role of electrostatic interactions in the dynamics of MscL gating.—Zhong, D., Blount, P. Electrostatics at the membrane define MscL channel mechanosensitivity and kinetics.

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.

Salmonella and Shigella bacteria require the type III secretion system (T3SS) to inject virulence proteins into their hosts and initiate infections. The tip proteins SipD and IpaD are critical components of the Salmonella and Shigella T3SS, respectively. Recently, SipD and IpaD have been shown to interact with bile salts, which are enriched in the intestines, and are hypothesized to act as environmental sensors for these enteric pathogens. Bile salts activate the Shigella T3SS but repress the Salmonella T3SS, and the mechanism of this differing response to bile salts is poorly understood. Further, how SipD binds to bile salts is currently unknown. Computer modeling predicted that IpaD binds the bile salt deoxycholate in a cleft formed by the N-terminal domain and the long central coiled coil of IpaD. Here, we used NMR methods to determine which SipD residues are affected by the interaction with the bile salts deoxycholate, chenodeoxycholate, and taurodeoxcholate. The bile salts perturbed nearly the same set of SipD residues; however, the largest chemical shift perturbations occurred away from what was predicted for the bile salt binding site in IpaD. Our NMR results indicate that that bile salt interaction of SipD will be different from what was predicted for IpaD, suggesting a possible mechanism for the differing response of Salmonella and Shigella to bile salts.

In the present study we identified a duck invariant chain (Ii) cDNA, named duck Ii-1, by RT-PCR and RACE. It was 1190 bp in length and contained a 669 bp open reading frame. An alternative transcript encoding a thyroglobulin (Tg)-containing form of Ii, named duck Ii-2, was also found in duck. The putative amino acid sequence of duck Ii-1 showed an 82% similarity to chicken Ii-1 and about 60% similarity to its mammalian homologues. The similarity of the Tg domain between duck and chicken Ii-2 was 96%, and about 70% between duck and mammalian Ii. The result of RT-PCR showed that Ii mRNA was extensively expressed in various tissues. High levels of both Ii-1 and Ii-2 mRNA were observed in the spleen and bursa of Fabricius. The predicted three-dimensional (3D) structures of duck Ii trimerization and Tg domain are similar to the corresponding regions of human Ii analyzed by comparative protein modeling. These findings indicate that the two isoforms of duck Ii, which strongly expressed in the major immune organs, share structural identity with human Ii.

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.

The bacterial mechanosensitive channel of large conductance (MscL) directly senses and responds to membrane tension. It serves as an "emergency release valve" upon acute decreases in the osmotic environment, thus preventing cell lysis. It is one of the best studied mechanosensitive channels and serves as a paradigm of how a channel senses and responds to its membrane environment. The MscL protein is highly conserved, found throughout the bacterial kingdom, and has been shown to encode a functional mechanosensitive channel in all species where it has been studied. However, channels from different species have shown some functional variance; an extreme example is the Mycobacterium tuberculosis MscL, which when heterologously expressed in Escherichia coli requires significantly more membrane tension for gating than the endogenous E. coli MscL. We previously speculated that the membrane environment or factors not found in E. coli promoted the proper gating of the M. tuberculosis MscL channel in its native environment. Here, by reconstituting the M. tuberculosis and E. coli MscL channels in various lipids, we demonstrate that inclusion of phosphatidylinositol, a lipid found in M. tuberculosis but not E. coli, is sufficient for gating of the M. tuberculosis MscL channel within a physiological range of membrane tension.

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is a homopentameric channel in the plasma membrane that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. It is one of the best studied mechanosensors and serves as a paradigm of how a protein can sense and respond to membrane tension. While all orthologues tested thus far encode channel activity, many show functional differences. For example, E coli MscL has more stable openings and is more mechanosensitive than S. aureus MscL. Here we have utilized a chimera approach to correlate structural features with functional differences. Surprisingly, the data indicate that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, plays a major role in both the open dwell time and mechanosensitivity of the channel for both orthologues. The features of this single residue and interactions of this site were studied by employing site directed mutagenesis, cysteine trapping, and single channel patch clamp recordings. In sum, the data suggest that this single residue acts as a multi-switch, interacting differentially with various regions of the pentameric complex as the channel gates.

The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65-82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded.

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.

Summary MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic "emergency release valve," is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E. coli I49 and the equivalent S. aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.