Metabolic and immune-sensitive contacts between lipid droplets and endoplasmic reticulum reconstituted in vitro.

Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD-ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Effect of Rab18 on liver injury and lipid accumulation by regulating perilipin 2 and peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is currently one of the most common chronic liver diseases worldwide, characterized by the presence of lipid droplets. Rab18 is an important lipid droplet protein; however, its effects and mechanisms of action on NAFLD remain unclear. METHODS: Free fatty acid-stimulated AML-12 cells and high-fat diet (HFD)-fed mice were used as NAFLD models. Lentiviruses overexpressing Rab18 (Rab18-OE) or knockdown (Rab18-KD) were used to generate stable cell lines for genetic analysis. Blood serum levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, and leptin were measured using a biochemical autoanalyzer. Hematoxylin and eosin staining was performed to detect pathological damage to the liver. Lipid accumulation in the cells was assessed by Oil Red O staining. Target expression was measured using qPCR, western blotting, and immunocytochemistry. RESULTS: Rab18 mRNA and protein expression levels increased in free fatty acid-stimulated AML-12 cells and the livers of HFD-fed mice. Rab18-OE increased lipid accumulation in vitro, which was attenuated by Rab18-KD. In vivo, Rab18-OE augmented liver pathological damage, serum alanine aminotransferase/aspartate aminotransferase activity, and triglyceride, total cholesterol, and low-density lipoprotein levels, whereas Rab18-KD decreased these indicators. Rab18-KD also downregulated blood glucose levels in HFD-fed mice. Mechanistically, Rab18-OE and Rab18-KD regulated the mRNA and protein expression levels of perilipin 2 (PLIN2) and peroxisome proliferator-activated receptor gamma (PPARγ) in vitro and in vivo, respectively. Immunocytochemistry revealed that Rab18 colocalized with PLIN2 and PPARγ in AML-12 cells. CONCLUSION: Rab18 expression was elevated in vitro and in vivo in the NAFLD mouse model. Rab18 regulates PLIN2 and PPARγ expression to exaggerate liver injury and lipid accumulation in patients with NAFLD. Thus, Rab18 may be a crucial protein in this disease and a potential therapeutic target.

Rab18 Drift in Lipid Droplet and Endoplasmic Reticulum Interactions of Adipocytes under Obesogenic Conditions.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER-LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER-LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER-LD connection and Rab18 distribution at ER-LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER-LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER-LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER-LD interactions and interfere with Rab18 trafficking through these contact sites.

Trappc9 Deficiency Impairs the Plasticity of Stem Cells.

Genetic mutations of trappc9 cause intellectual disability with the atrophy of brain structures and variable obesity by poorly understood mechanisms. Trappc9-deficient mice develop phenotypes resembling pathological changes in humans and appear overweight shortly after weaning, and thus are useful for studying the pathogenesis of obesity. Here, we investigated the effects of trappc9 deficiency on the proliferation and differentiation capacity of adipose-derived stem cells (ASCs). We isolated ASCs from mice before overweight was developed and found that trappc9-null ASCs exhibited signs of premature senescence and cell death. While the lineage commitment was retained, trappc9-null ASCs preferred adipogenic differentiation. We observed a profound accumulation of lipid droplets in adipogenic cells derived from trappc9-deficient ASCs and marked differences in the distribution patterns and levels of calcium deposited in osteoblasts obtained from trappc9-null ASCs. Biochemical studies revealed that trappc9 deficiency resulted in an upregulated expression of rab1, rab11, and rab18, and agitated autophagy in ASCs. Moreover, we found that the content of neural stem cells in both the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus vastly declined in trappc9-null mice. Collectively, our results suggest that obesity, as well as brain structure hypoplasia induced by the deficiency of trappc9, involves an impairment in the plasticity of stem cells.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association.

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

Rab18: new insights into the function of an essential protein.

Rab18 is one of the small number of conserved Rab proteins which have been traced to the last eukaryotic common ancestor. It is found in organisms ranging from humans to trypanosomes, and localizes to multiple organelles, including most notably endoplasmic reticulum and lipid droplets. In humans, absence of Rab18 leads to a severe illness known as Warburg-Micro syndrome. Despite this evidence that Rab18 is essential, its role in cells remains mysterious. However, recent studies identifying effectors and interactors of Rab18, are now shedding light on its mechanism of action, suggesting functions related to organelle tethering and to autophagy. In this review, we examine the variety of roles proposed for Rab18 with a focus on new evidence giving insights into the molecular mechanisms it utilizes. Based on this summary of our current understanding, we identify priority areas for further research.

Rab18 regulates lipolysis via Arf/GBF1 and adipose triglyceride lipase.

Rab18 is a small GTPase associated with lipid droplets and other membranes. While it likely has multiple functions on lipid droplets, one proposed function is regulation of lipolysis. Previous work has concentrated on regulation of autophagy; however, in this study, we provide evidence that Rab18 plays a role upstream of the cytosolic lipolytic enzyme adipose triglyceride lipase (ATGL) and that recruitment of ATGL by Rab18 is mediated by elements of the Arf/GBF1 machinery. We find that Arf4-GFP is accumulated on the subset of lipid droplets associated with Rab18, and that this association is lost within 5 min upon treatment with 5 µg/ml of the drug brefeldin A, which targets GBF1 and other Sec7-domain containing Arf exchange factors. ATGL-GFP is also recruited to lipid droplets, but is lost more slowly after treatment with 5 µg/ml brefeldin A, with significant loss from lipid droplets after 1 h treatment, and almost complete loss from lipid droplets 4 h after brefeldin A treatment. Upon overexpression of the dominant negative GDP-locked cerulean-Rab18-S22 N, GFP-ATGL and Arf4 are lost from the surface of lipid droplets similarly to BFA treatment. This study establishes, for the first time, an essential role for Rab18 in recruiting ATGL to lipid droplets.

DFCP1 associates with lipid droplets.

Double FYVE-containing protein 1 (DFCP1) is ubiquitously expressed, participates in intracellular membrane trafficking and labels omegasomes through specific interactions with phosphatidylinositol-3-phosphate (PI3P). Previous studies showed that subcellular DFCP1 proteins display multi-organelle localization, including in the endoplasmic reticulum (ER), Golgi apparatus and mitochondria. However, its localization and function on lipid droplets (LDs) remain unclear. Here, we demonstrate that DFCP1 localizes to the LD upon oleic acid incubation. The ER-targeted domain of DFCP1 is indispensable for its LD localization, which is further enhanced by double FYVE domains. Inhibition of PI3P binding at the FYVE domain through wortmannin treatment or double mutation at C654S and C770S have no effect on DFCP1's LD localization. These show that the mechanisms for DFCP1 targeting the omegasome and LDs are different. DFCP1 deficiency in MEF cells causes an increase in LD number and reduces LD size. Interestingly, DFCP1 interacts with GTP-bound Rab18, an LD-associated protein. Taken together, our work demonstrates the dynamic localization of DFCP1 is regulated by nutritional status in response to cellular metabolism.

Cul3 neddylation is crucial for gradual lipid droplet formation during adipogenesis.

Cullin 3 (Cul3) belongs to the family of cullins (Cul1-7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.

Small GTPases in peroxisome dynamics.

In this review article, we summarize current knowledge on peroxisome biogenesis/functions and the role that small GTPases may play in these processes. Precise intracellular distribution of cell organelles requires their regulated association to microtubules and the actin cytoskeleton. In this respect, RhoGDP/RhoGTP favor binding of peroxisomes to microtubules and actin filaments. In its GTP-bound form, RhoA activates a regulatory cascade involving Rho kinaseII and non-muscle myosinIIA. Such interactions frequently depend on phosphoinositides (PIs) of which PI4P, PI(4,5)P2, and PI(3,5)P2 were found to be present in the peroxisomal membrane. PIs are pivotal determinants of intracellular signaling and known to regulate a wide range of cellular functions. In many of these functions, small GTPases are implicated. The small GTPase ADP-ribosylation factor 1 (Arf1), for example, is known to stimulate synthesis of PI4P and PI(4,5)P2 on the Golgi to regulate protein and lipid sorting. In vitro binding assays localized Arf1 and the COPI complex to peroxisomes. In light of the recent discussion of pre-peroxisomal vesicle generation at the ER, peroxisomal Arf1-COPI vesicles may serve retrograde transport of ER-resident components. A mass spectrometric screen localized various Rab proteins to peroxisomes. Overexpression of these proteins in combination with laser-scanning fluorescence microscopy co-localized Rab6, Rab8, Rab10, Rab14, and Rab18 with peroxisomal structures. By analogy to the role these proteins play in other organelle dynamics, we may envisage what the function of these proteins may be in relation to the peroxisomal compartment.

The RAB GTPase RAB18 modulates macroautophagy and proteostasis.

Macroautophagy is a conserved degradative pathway and its deterioration is linked to disturbances in cellular proteostasis and multiple diseases. Here, we show that the RAB GTPase RAB18 modulates autophagy in primary human fibroblasts. The knockdown of RAB18 results in a decreased autophagic activity, while its overexpression enhances the degradative pathway. Importantly, this function of RAB18 is dependent on RAB3GAP1 and RAB3GAP2, which might act as RAB GEFs and stimulate the activity of the RAB GTPase. Moreover, the knockdown of RAB18 deteriorates proteostasis and results in the intracellular accumulation of ubiquitinated degradation-prone proteins. Thus, the RAB GTPase RAB18 is a positive modulator of autophagy and is relevant for the maintenance of cellular proteostasis.

Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus).

The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

Bacterial-induced expression of RAB18 protein in Orzya sativa salinity stress and insights into molecular interaction with GTP ligand.

In the present research, we have studied the inoculation effects of two root-associated plant growth-promoting rhizobacteria (PGPR) in rice and provide the pieces of evidence that the inoculation of the PGPR could potentially result in inducing the expression of the salt stress-related RAB18 plant gene under varying degrees of salinity stress. The sequenced putative gene of RAB18 of Oryza sativa in this study is 740 bp long, has a content of 44.4%, and a molecular weight of 492 102.00 Da. BLAST homology patterns revealed sequence similarity with the previously sequenced RAB in model plant species. We demonstrate the mode of action of this stress-related protein by performing comparative modeling of Q10RT8 (Os03g0146000 protein, homolog of the sequenced RAB18; O. sativa subsp. japonica) using energy minimization, molecular dynamic simulations, and molecular docking of a guanosine triphosphate (GTP) ligand with the protein. The docking results indicated that Ser21, Ala22, Lys25, Asp68, Ala70, Glu73, and Arg74 are important determinant residues for functional interaction with the GTP ligand. The present research contributes to the understanding of the PGPR inoculation in salinity stress. Additionally, it provides the layout of the understanding of the molecular interactions between RAB and GTP ligand.

Spirulina supplement and exercise training affect lipid droplets-related genes expression in visceral adipose tissue.

Objective: Disruption of lipid droplets (LDs) is associated with many metabolic diseases. Spirulina, as a natural bioactive dietary supplement, along with exercise training, may improve lipid metabolism; however, their effects on LDs-regulated genes in visceral adipose tissue are still unclear. This study aimed to investigate the effects of six-week Spirulina supplementation along with exercise training on LDs regulating gene expression. Materials and Methods: Fifty-six male Wistar rats were divided into six groups: saline (control), control+Spirulina (Spirulina), aerobic interval training (AIT), AIT+ Spirulina (AIT+Spirulina), resistance training and resistance+ Spirulina. The supplement groups consumed 500 mg/kg Spirulina five days per week. The training groups performed AIT (5 times per week) and resistance training (3 times per week) for 6 weeks. LDs regulating genes expression in visceral adipose tissue (Zw10, Bscl2, DFCP1, Rab18, Syntaxin 18, Acsl3, and Plin2) was analyzed by real-time PCR. Results: Spirulina and exercise training had no significant effects on the gene expression of Syntaxin18 (p=0.69) and DFCP1 (p=0. 84), ACSL3 (p=0.98), or BSCL2 (p=0.58). In addition, Spirulina was found to significantly attenuate the expression of Plin2 (p=0.01) and Rab18 (p=0.01) genes compared to the control, AIT, and resistance training groups. However, Plin2 gene expression was higher in the resistance training than the AIT. Furthermore, Spirulina decreased ZW10 (p=0.03) gene expression in visceral adipose tissue compared to the control, AIT, and resistance training groups. Unexpectedly, Spirulina supplementation decreased the expression of these genes even more when taken without exercise training. Conclusion: Spirulina supplementation and exercise training have significant effects on LDs-regulated genes in visceral adipose tissue.

Growth, fusion and degradation of lipid droplets: advances in lipid droplet regulatory protein.

Context: An adequate supply of energy is essential for the proper functioning of all life activities in living organisms. As organelles that store neutral lipids, lipid droplets (LDs) are involved in the synthesis and metabolism of lipids in cells and are also an important source of energy supply.Methods and mechanisms: A comprehensive summary of the literature was first carried out to screen for relevant proteins affecting the morphological size of LDs.The size of milk fat globules (MFGs) is directly influenced by the morphological size of LDs, which also controls the energy storage capacity of LDs. In this review, we detail the progress of research into the role of some protein in regulating the morphological size of LDs.Conclusion: It has been discovered that the number of protein are involved in the control of LD growth and degradation, such as Rab18-mediated local synthesis of triacylglycerol (TAG), cell death-inducing DFF45-like effector family proteins (CIDEs)-mediated atypical fusion between LDs, Stomatin protein-mediated LD fusion and autophagy-related proteins (ATGs)-mediated autophagic degradation of LDs. However, more studies are needed in the future to enrich the network of mechanisms that regulate the morphological size of LDs.

Rab18 maintains homeostasis of subcutaneous adipose tissue to prevent obesity-induced metabolic disorders.

Metabolically healthy obesity refers to obese individuals who do not develop metabolic disorders. These people store fat in subcutaneous adipose tissue (SAT) rather than in visceral adipose tissue (VAT). However, the molecules participating in this specific scenario remain elusive. Rab18, a lipid droplet (LD)-associated protein, mediates the contact between the endoplasmic reticulum (ER) and LDs to facilitate LD growth and maturation. In the present study, we show that the protein level of Rab18 is specifically upregulated in the SAT of obese people and mice. Rab18 adipocyte-specific knockout (Rab18 AKO) mice had a decreased volume ratio of SAT to VAT compared with wildtype mice. When subjected to high-fat diet (HFD), Rab18 AKO mice had increased ER stress and inflammation, reduced adiponectin, and decreased triacylglycerol (TAG) accumulation in SAT. In contrast, TAG accumulation in VAT, brown adipose tissue (BAT) or liver of Rab18 AKO mice had a moderate increase without ER stress stimulation. Rab18 AKO mice developed insulin resistance and systematic inflammation. Rab18 AKO mice maintained body temperature in response to acute and chronic cold induction with a thermogenic SAT, similar to the counterpart mice. Furthermore, Rab18-deficient 3T3-L1 adipocytes were more prone to palmitate-induced ER stress, indicating the involvement of Rab18 in alleviating lipid toxicity. Rab18 AKO mice provide a good animal model to investigate metabolic disorders such as impaired SAT. In conclusion, our studies reveal that Rab18 is a key and specific regulator that maintains the proper functions of SAT by alleviating lipid-induced ER stress.

Voltage-dependent anion channel 3 (VDAC3) mediates P. syringae induced ABA-SA signaling crosstalk in Arabidopsis thaliana.

Pathogen severely affects plant mitochondrial processes including respiration, however, the roles and mechanism of mitochondrial protein during the immune response remain largely unexplored. The interplay of plant hormone signaling during defense is an outcome of plant pathogen interaction. We recently discovered that the Arabidopsis calcineurin B-like interacting protein kinase 9 (AtCIPK9) interacts with the voltage-dependent anion channel 3 (AtVDAC3) and inhibits MV-induced oxidative damage. Here we report the characterization of AtVDAC3 in an antagonistic interaction pathway between abscisic acid (ABA) and salicylic acid (SA) signaling in Pseudomonas syringae -Arabidopsis interaction. In this study, we observed that mutants of AtVDAC3 were highly susceptible to Pseudomonas syringae infection as compared to the wild type (WT) Arabidopsis plants. Transcripts of VDAC3 and CIPK9 were inducible upon ABA application. Following pathogen exposure, expression analyses of ABA and SA biosynthesis genes indicated that the function of VDAC3 is required for isochorisimate synthase 1 (ICS1) expression but not for Nine-cis-epoxycaotenoid dioxygenase 3 (NCED3) expression. Despite the fact that vdac3 mutants had increased NCED3 expression in response to pathogen challenge, transcripts of ABA sensitive genes such as AtRD22 and AtRAB18 were downregulated even after exogenous ABA application. VDAC3 is required for ABA responsive genes expression upon exogenous ABA application. We also found that Pseudomonas syringae-induced SA signaling is downregulated in vdac3 mutants since overexpression of VDAC3 resulted in hyperaccumulation of Pathogenesis related gene1 (PR1) transcript. Interestingly, ABA application prior to P. syringae inoculation resulted in the upregulation of ABA responsive genes like Responsive to ABA18 (RAB18) and Responsive to dehydration 22 (RD22). Intriguingly, in the absence of AtVDAC3, Pst challenge can dramatically increase ABA-induced RD22 and RAB18 expression. Altogether our results reveal a novel Pathogen-SA-ABA interaction pathway in plants. Our findings show that ABA plays a significant role in modifying plant-pathogen interactions, owing to cross-talk with the biotic stress signaling pathways of ABA and SA.

PheLBD29, an LBD transcription factor from Moso bamboo, causes leaf curvature and enhances tolerance to drought stress in transgenic Arabidopsis.

The lateral organ boundaries domain (LBD), a unique family of transcription factors in higher plants, plays a key role in plant growth and development, and stress response. Here, we report on the novel lateral organ boundaries domain (LBD) gene PheLBD29, a nuclear localization protein that can bind the conserved GCCCCG sequence, as determined by electrophoretic mobility shift assay (EMSA). PheLBD29 was highly expressed in blade leaf and significantly induced by polyethylene glycol (PEG). Overexpression of PheLBD29 leads to small and abaxially rolled leaves in Arabidopsis, and anatomically, 35S:PheLBD29 Arabidopsis leaves showed transformation of adaxial cells into abaxial cells. Moreover, overexpression of PheLBD29 in Arabidopsis increased plant tolerance to drought stress, by accumulation of more soluble sugars, less malondialdehyde (MDA), and had lower REL levels under drought stress. Transient expression assay revealed PheLBD29 directly bound to the promoter region of RAB18. In addition, 35S:PheLBD29 Arabidopsis showed higher sensitivity to abscisic acid (ABA) than the wild type. Therefore, we conclude that PheLBD29 may participate in the ABA-dependent signaling pathway to improve drought tolerance. Our study provides new evidence for a Moso bamboo LBD protein regulatory module in leaf curvature and drought resistance.

Exome sequencing identifies a novel pathogenic variant in RAB3GAP1 causing Warburg Micro syndrome in a Pakistani family.

BACKGROUND: Warburg Micro (WARBM) syndrome is a rare heterogeneous recessive genetic disorder characterized by ocular, neurological, and endocrine problems. To date, disease-causing variants in four genes have been identified to cause this syndrome; of these, RAB3GAP1 variants are the most frequent. Very little is known about WARBM syndrome in rural populations. OBJECTIVES: This study aims to investigate the genetics underpinnings of WARBM syndrome in a Pashtun family with two patients from Pakistan. The patients presented with spastic diplegia, severe intellectual disability, microphthalmia, microcornea, congenital cataracts, optic atrophy, and hypogonadism. METHODS: Magnetic resonance imaging (MRI) analysis revealed pronounced cerebral atrophy including corpus callosum hypoplasia and polymicrogyria. Exome sequencing and subsequent filtering identified a novel homozygous missense variant NM_001172435: c.2891A>G, p.Gln964Arg in the RAB3GAP1 gene. The variant was validated, and its segregation confirmed, by Sanger sequencing. RESULTS: Multiple prediction tools assess this variant to be damaging, and structural analysis of the protein shows that the mutant amino acid residue affects polar contact with the neighboring atoms. It is extremely rare and is absent in all the public databases. Taken together, these observations suggest that this variant underlies Micro syndrome in our family and is extremely important for management and family planning. CONCLUSIONS: Identification of this extremely rare variant extends the mutations spectrum of Micro syndrome. Screening more families, especially in underrepresented populations, will help unveil the mutation spectrum underlying this syndrome.