Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.     

Selective Impairment of a Subset of Ran-GTP-binding Domains of Ran-binding Protein 2 (Ranbp2) Suffices to Recapitulate the Degeneration of the Retinal Pigment Epithelium (RPE) Triggered by Ranbp2 Ablation

Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2^−/−, with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2^−/− background. Among the transgenic lines produced, only Tg^RBD2/3*-HA::RPE-cre::Ranbp2^−/−-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2^−/−. By contrast, Tg^RBD2/3*-HA expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2^−/− and Tg^RBD2/3*-HA::RPE-cre::Ranbp2^−/− share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.     

Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in Vivo due to Impaired Signal Regulatory Protein α (SIRPα) Signaling

Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1α,25(OH)₂-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47^−/− mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)⁺ osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor κβ ligand) was reduced in CD47^−/− BMC, as compared with CD47^+/+ BMC. The stromal cell phenotype in CD47^−/− BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and α-1-collagen, and reduced mineral deposition, as compared with that in CD47^+/+ BMC. CD47 is a ligand for SIRPα (signal regulatory protein α), which showed strongly reduced tyrosine phosphorylation in CD47^−/− bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRPα cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47^−/− and non-signaling SIRPα mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47^−/− mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

Vacuolar-Iron-Transporter1-Like Proteins Mediate Iron Homeostasis in Arabidopsis

Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe) storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1). Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140), AtVTL2 (At1g76800) or AtVTL5 (At3g25190) in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.     

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition,Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6^−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6^−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6^−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.     

Disassembly of All SNARE Complexes by N-Ethylmaleimide-sensitive Factor (NSF) Is Initiated by a Conserved 1:1 Interaction between α-Soluble NSF Attachment Protein (SNAP) and SNARE Complex

Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.     

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO⁻₃)) residues by treatment with bisulfite. The poly(U(SO⁻₃)₂, C₃) and poly(U(SO⁻₃)₉, C₁) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO⁻₃) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO⁻₃) residues into U.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2^−/−) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2^−/− macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.     

Pdsg1 and Pdsg2, Novel Proteins Involved in Developmental Genome Remodelling in Paramecium

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.     

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan^R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.     

Therapeutic Benefits of Induced Pluripotent Stem Cells in Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive increases in vascular resistance and the remodeling of pulmonary arteries. The accumulation of inflammatory cells in the lung and elevated levels of inflammatory cytokines in the bloodstream suggest that inflammation may play a role in PAH. In this study, the benefits of induced pluripotent stem cells (iPSCs) and iPSC-conditioned medium (iPSC CM) were explored in monocrotaline (MCT)-induced PAH rats. We demonstrated that both iPSCs and iPSC CM significantly reduced the right ventricular systolic pressure and ameliorated the hypertrophy of the right ventricle in MCT-induced PAH rats in models of both disease prevention and disease reversal. In the prevention of MCT-induced PAH, iPSC-based therapy led to the decreased accumulation of inflammatory cells and down-regulated the expression of the IL-1β, IL-6, IL-12α, IL-12β, IL-23 and IFNγ genes in lung specimens, which implied that iPSC-based therapy may be involved in the regulation of inflammation. NF-κB signaling is essential to the inflammatory cascade, which is activated via the phosphorylation of the NF-κB molecule. Using the chemical inhibitor specifically blocked the phosphorylation of NF-κB, and in vitro assays of cultured human M1 macrophages implied that the anti-inflammation effect of iPSC-based therapy may contribute to the disturbance of NF-κB activation. Here, we showed that iPSC-based therapy could restore the hemodynamic function of right ventricle with benefits for preventing the ongoing inflammation in the lungs of MCT-induced PAH rats by regulating NF-κB phosphorylation.     

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.     

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome

Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, α-carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within α-carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes. It is present in all α-carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an α-carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3-Å resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the α-carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here α-carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to α-carboxysome function.     

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Calcium (Ca²⁺) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K_d ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca²⁺/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.