Bacterial Alarmone,Guanosine 5′‐Diphosphate 3′‐Diphosphate (ppGpp), Predominantly Binds the β′ Subunit of Plastid‐Encoded Plastid RNA Polymerase in Chloroplasts

It's alarming : Bacterial alarmone guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp), which is a key regulatory molecule that controls the stringent response, also exists in chloroplasts of plant cells. Cross‐linking experiments with 6‐thioguanosine 5′‐diphosphate 3′‐diphosphate (6‐thioppGpp) and chloroplast RNA polymerase indicate that ppGpp binds the β′ subunit of plastid‐encoded plastid RNA polymerase that corresponds to the Escherichia coli β′ subunit.

     

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1β

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP₃ generation, which results in intracellular Ca²⁺ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca²⁺ release-activated Ca²⁺ (CRAC) current and, in association with TRPC1, the store-operated Ca²⁺ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca²⁺ channel (ARC/LRC), store-independent Ca²⁺ influx activated by the secretory pathway Ca²⁺-ATPase (SPCA2) and the small conductance Ca²⁺-activated K⁺ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca²⁺ signals triggered by each variant, and their downstream modulatory effect within the cell.     

The α2δ Calcium Channel Subunit Accessorily and Independently Affects the Biological Function of Ditylenchus destructor

The α₂δ subunit is a high-voltage activated (HVA) calcium channel (Ca_v1 and Ca_v2) auxiliary subunit that increases the density and function of HVA calcium channels in the plasma membrane of mammals. However, its function in plant parasitic nematodes remains unknown. In this study, we cloned the full-length cDNA sequence of the voltage-gated calcium channel (VGCC) α₂δ subunit (named DdCa_vα₂δ) in Ditylenchus destructor. We found that DdCa_vα₂δ tends to be expressed in the egg stage, followed by the J3 stage. RNA-DIG in situ hybridization experiments showed that the DdCa_vα₂δ subunit was expressed in the body wall, esophageal gland, uterus, post uterine, and spicules of D. destructor. The in vitro application of RNA interference (RNAi) affected the motility, reproduction, chemotaxis, stylet thrusting, and protein secretion of D. destructor to different degrees by targeting DdCα1D, DdCα1A, and DdCa_vα₂δ in J3 stages, respectively. Based on the results of RNAi experiments, it was hypothesized that L-type VGCC may affect the motility, chemotaxis, and stylet thrusting of D. destructor. Non-L-type VGCC may affect the protein secretion and reproduction of D. destructor. The DdCa_vα₂δ subunit gene also affected the motility, chemotaxis, and reproduction of D. destructor. These findings reveal the independent function of the VGCC α₂δ subunit in D. destructor as well as give a theoretical foundation for future research on plant parasitic nematode VGCC.     

Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.     

αN-Acetyl β-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers

The opioid peptide β-endorphin coexists in the pituitary and brain in its ^αN-acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, ^αN-acetyl β-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of N-methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, ^αN-acetyl β-Endorphin reduced the analgesia of morphine, β-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by β-Endorphin and absent in σ1R^−/− mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, β-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the ^αN-acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological β-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that β-Endorphin and ^αN-acetyl β-Endorphin are endogenous ligands of σ1R.     

Plastid Anionic Lipids Are Essential for the Development of Both Photosynthetic and Non-Photosynthetic Organs in Arabidopsis thaliana

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.     

KOMPEITO,an Atypical Arabidopsis Rhomboid-Related Gene,Is Required for Callose Accumulation and Pollen Wall Development

Fertilization is a key event for sexually reproducing plants. Pollen–stigma adhesion, which is the first step in male–female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a β-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen–stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.     

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions

The α₂δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α₂δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α₂δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α₂δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α₂δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α₂δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α₂δ-1-3 isoforms (α₂δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α₂δ proteins. In contrast to this redundant presynaptic function, α₂δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α₁-α₂δ protein–protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α₂δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α₂δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α₂δ and Cachd1.     

Pathogenic Impact of α-Synuclein Phosphorylation and Its Kinases in α-Synucleinopathies

α-Synuclein is a protein with a molecular weight of 14.5 kDa and consists of 140 amino acids encoded by the SNCA gene. Missense mutations and gene duplications in the SNCA gene cause hereditary Parkinson’s disease. Highly phosphorylated and abnormally aggregated α-synuclein is a major component of Lewy bodies found in neuronal cells of patients with sporadic Parkinson’s disease, dementia with Lewy bodies, and glial cytoplasmic inclusion bodies in oligodendrocytes with multiple system atrophy. Aggregated α-synuclein is cytotoxic and plays a central role in the pathogenesis of the above-mentioned synucleinopathies. In a healthy brain, most α-synuclein is unphosphorylated; however, more than 90% of abnormally aggregated α-synuclein in Lewy bodies of patients with Parkinson’s disease is phosphorylated at Ser129, which is presumed to be of pathological significance. Several kinases catalyze Ser129 phosphorylation, but the role of phosphorylation enzymes in disease pathogenesis and their relationship to cellular toxicity from phosphorylation are not fully understood in α-synucleinopathy. Consequently, this review focuses on the pathogenic impact of α-synuclein phosphorylation and its kinases during the neurodegeneration process in α-synucleinopathy.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.