Steroidogenic Acute Regulatory Protein-related Lipid Research Articles

Sterol regulatory element binding transcription factor 1 promotes proliferation and migration in head and neck squamous cell carcinoma.

Sterol-regulatory element-binding protein 1 (SREBP1) is a transcription factor involved in lipid metabolism that is encoded by sterol regulatory element binding transcription factor 1(SREBF1). SREBP1 overexpression is associated with the progression of several human tumors; however, the role of SREBP1 in head and neck squamous cell carcinoma (HNSC) remains unclear. SREBF1 expression in pan-cancer was analyzed using the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data, and the association between SREBF1 expression and clinical characteristics of HNSC patients was examined using the UALCAN database. Enrichment analysis of SREBF1-related genes was performed using the Cluster Profiler R package. TCGA database was used to investigate the relationship between immune cell infiltration and SREBF1 expression. CCK-8, flow cytometry, and wound healing assays were performed to investigate the effect of SREBF1 knockdown on the proliferation and migration of HNSC cells. SREBF1 was significantly upregulated in several tumor tissues, including HNSC, and SREBF1 overexpression was positively correlated with sample type, cancer stage, tumor grade, and lymph node stage in HNSC patients. Gene enrichment analysis revealed that SREBF1 is associated with DNA replication and homologous recombination. SREBF1 upregulation was positively correlated with the infiltration of cytotoxic cells, B cells, T cells, T helper cells, and NK CD56 bright cells in HNSC. Knockdown of SREBF1 inhibited the proliferation and migration of HNSC cells (Hep2 and TU212) and induced apoptosis by downregulating the expression of steroidogenic acute regulatory protein-related lipid transfer 4 (STARD4). SREBF1 may promote HNSC proliferation, migration and inhibit apoptosis by upregulating STARD4 and affecting the level of immune cell infiltration.

A conserved mechanism determines the activity of two pivotal transcription factors that control epidermal cell differentiation in Arabidopsis thaliana

The surface of plants is covered by the epidermis, which protects the plant’s body from the external environment and mediates inter-cell layer signaling to regulate plant development. Therefore, the manifestation of epidermal traits at a precise location is a prerequisite for their normal growth and development. In Arabidopsis thaliana, class IV homeodomain-leucine zipper transcription factors PROTODERMAL FACTOR2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1) play redundant roles in epidermal cell differentiation. Nevertheless, several pieces of evidence suggest that the activity and/or function of PDF2 and ATML1 are regulated differently. The role of the steroidogenic acute regulatory protein-related lipid transfer (START) domain of ATML1 in restricting this protein’s activity has been demonstrated; however, whether this lipid-dependent mechanism regulates PDF2 expression is unknown. In this study, we demonstrated that the START domains of PDF2 and ATML1, regulate protein turnover in a position-dependent manner and affect the dimeric proteins. Our results show that a conserved mechanism provides the basis for the functional redundancy of PDF2 and ATML1 in epidermal cell differentiation and that an unidentified regulatory layer specific to PDF2 or ATML1 is responsible for the difference in the activity and/or function of PDF2 and ATML1.

Characterization of promoter elements of isoprene-responsive genes and the ability of isoprene to bind START domain transcription factors.

Isoprene has recently been proposed to be a signaling molecule that can enhance tolerance of both biotic and abiotic stress. Not all plants make isoprene, but all plants tested to date respond to isoprene. We hypothesized that isoprene interacts with existing signaling pathways rather than requiring novel mechanisms for its effect on plants. We analyzed the cis-regulatory elements (CREs) in promoters of isoprene-responsive genes and the corresponding transcription factors binding these promoter elements to obtain clues about the transcription factors and other proteins involved in isoprene signaling. Promoter regions of isoprene-responsive genes were characterized using the Arabidopsis cis-regulatory element database. CREs bind ARR1, Dof, DPBF, bHLH112, GATA factors, GT-1, MYB, and WRKY transcription factors, and light-responsive elements were overrepresented in promoters of isoprene-responsive genes; CBF-, HSF-, WUS-binding motifs were underrepresented. Transcription factors corresponding to CREs overrepresented in promoters of isoprene-responsive genes were mainly those important for stress responses: drought-, salt/osmotic-, oxidative-, herbivory/wounding and pathogen-stress. More than half of the isoprene-responsive genes contained at least one binding site for TFs of the class IV (homeodomain leucine zipper) HD-ZIP family, such as GL2, ATML1, PDF2, HDG11, ATHB17. While the HD-zipper-loop-zipper (ZLZ) domain binds to the L1 box of the promoter region, a special domain called the steroidogenic acute regulatory protein-related lipid transfer, or START domain, can bind ligands such as fatty acids (e.g., linolenic and linoleic acid). We tested whether isoprene might bind in such a START domain. Molecular simulations and modeling to test interactions between isoprene and a class IV HD-ZIP family START-domain-containing protein were carried out. Without membrane penetration by the HDG11 START domain, isoprene within the lipid bilayer was inaccessible to this domain, preventing protein interactions with membrane bound isoprene. The cross-talk between isoprene-mediated signaling and other growth regulator and stress signaling pathways, in terms of common CREs and transcription factors could enhance the stability of the isoprene emission trait when it evolves in a plant but so far it has not been possible to say what how isoprene is sensed to initiate signaling responses.

Mechanisms of Nonvesicular Ceramide Transport.

Ceramides, as key components of cellular membranes, play essential roles in various cellular processes, including apoptosis, cell proliferation, and cell signaling. Ceramides are the precursors of all complex sphingolipids in eukaryotic cells. They are synthesized in the endoplasmic reticulum and are further processed at the Golgi apparatus. Therefore, ceramides have to be transported between these two organelles. In mammalian cells, the ceramide transfer protein forms a contact site between the ER and the trans-Golgi region and transports ceramide utilizing its steroidogenic acute regulatory protein-related lipid transfer domain. In yeast, multiple mechanisms of nonvesicular ceramide transport have been described. This involves the nuclear-vacuolar junction protein Nvj2, the yeast tricalbin proteins, and the lipocalin-like protein Svf1. This review aims to provide a comprehensive overview of nonvesicular ceramide transport mechanisms and their relevance in cellular physiology. We will highlight the physiological and pathological consequences of perturbations in nonvesicular ceramide transport and discuss future challenges in identifying and analyzing ceramide transfer proteins.

Evolutionary relationship analysis of STARD gene family and VvSTARD5 improves tolerance of salt stress in transgenic tomatoes.

The steroidogenic acute regulatory protein-related lipid transfer domain (STARD) forms a protein that can bind membrane-derived phospholipid second messengers and plasma membranes. Although it has been reported in many plants, the evolutionary relationship of the STARD gene family has not been systematically analyzed, and functions of the HD-START and HD-START-MEKHLA domain subgroup genes under hormone and abiotic stress are also unclear in grapes. This study identified and analyzed 23 VvSTARD genes, which were distinctly divided into five subgroups according to five conserved domain types. The analyses of codon preference, selective pressure, and synteny relationship revealed that grape had higher homology with Arabidopsis compared with rice. Interestingly, the expression levels of VvSTARD genes in subgroups 1, 2, and 3 exhibited significant upregulation under NaCl treatment at 24 h, but VvSTARD genes in subgroups 4 and 5 were upregulated under methyl jasmonate (MeJA) treatment at 24 h. The subcellular localization showed that VvSTARD5 was localized in the nucleus. Additionally, under NaCl treatment at 24 h, there were an obvious decrease in the relative electrical leakages and the content of malondialdehyde (MDA), while the relative expression level of VvSTARD5 and content of proline were obviously enhanced in three transgenic lines. Therefore, the overexpression of VvSTARD5 greatly increased the salt tolerance of transgenic tomatoes. Collectively, this study preliminarily explores the comprehensive function of the STARD gene family in grapes and verifies the function of VvSTARD5 in response to salt.

In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum–resident vesicle-associated membrane protein–associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein–related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

The significance of StAR-related lipid transfer protein-3 expression in breast cancer

StAR-related lipid transfer domain containing proteins (STARD3) are agroup of proteins that contain asteroidogenic acute regulatory protein-related lipid transfer domain. Breast cancer (BC) has been linked to the STARD3 gene. In this study, we sought to confirm the relationship of STARD3 protein expression with clinicopathological characteristics and BC molecular subtypes. Using tissue microarray, we examined the STARD3 protein expression in 200 BC tissues and 20 normal breast tissues. Higher protein expression of STARD3 was found in tumor tissues than normal tissues. One hundred and fifty-two (69.1%) of the 200 cases tested positive for STARD3 (high H-score), while seventy (30.9%) had alow STARD3 H-score. When STARD3 is present, its expression ranges from mild to strong. STARD3 H-score was strongly linked to human epidermal growth factor receptor 2 (HER2)-positive (p < 0.001) and estrogen receptor (ER)-positive (p < 0.009) patients, but not to triple-negative BC patients. STARD3 high H-score was associated with histological grade and tumor size. No significant associations were found with stage of the disease, pathological stage or node status. Our research revealed that STARD3 levels were higher in tissues from malignant BC, and it was associated with HER2 and ER, suggesting that it might be utilized as amarker for BC.

Identification of a key gene StAR-like-3 responsible for carotenoids accumulation in the noble scallop Chlamys nobilis

Carotenoids play important roles in living organisms. However, animals cannot synthesize carotenoids by themselves, and they must absorb and accumulate carotenoids from their diets in which some key genes are involved. In present study, a gene named StAR-like-3 was characterized in the noble scallop Chlamys nobilis, and its function was identified using golden scallops with higher carotenoids content and brown scallops with less carotenoids content by immunohistochemistry, carotenoid binding assay and RNAi. Results showed that the StAR-like-3 encodes a 54.7 kDa transmembrane protein (named as StAR3) of 481 amino acids containing a MENTAL domain and a START (Steroidogenic acute regulatory protein-related lipid transfer) domain, and its expression level in hemocytes and intestine of golden scallops were significantly higher than those of brown ones. Subsequently, the StAR3 protein was detected in the intestinal epithelial cells of golden scallops, and recombinant StAR3 could bind lutein conjugated to protein G and antibody to form a yellow complex, suggesting it is a carotenoid binding protein involving in carotenoids accumulation in golden scallops. Furthermore, total carotenoids content of hemolymph in golden scallops was significantly decreased when the expression of StAR-like-3 suppressed, suggesting this gene plays an important role in transport of carotenoids. Conclusion, the present results indicated that the StAR-like-3 is a key gene responsible for the carotenoids accumulation in the scallop.

Steroidogenic acute regulatory protein-related lipid transfer 4 (StarD4) promotes breast cancer cell proliferation and its mechanism

Oncogene StarD4 had the function of promoting proliferation and metastasis of triple-negative breast cancer (TNBC), but its clinical value and molecular mechanism are unknown. This paper found that StarD4 was highly expressed in cancer tissues of TNBC patients, and higher expression level of StarD4 in TNBC patient resulted in poorer prognosis. Based on transcriptomics of MDA-MB-231 cell model, the results of bioinformatics analysis showed that down-regulated expression level of StarD4 led to overall downregulation of cholesterol-relative genes and significant enrichment of cancer mechanism and pathway. Further analysis and investigation verified that StarD4 might cross-promote the protein stability of receptor ITGA5 through the cholesterol pathway to enhance TNBC progression, which provides guidance for clinical application of TNBC diagnosis and treatment.

The lipid-binding START domain regulates the dimerization of ATML1 via modulating the ZIP motif activity in Arabidopsis thaliana.

In Arabidopsis thaliana, the epidermis is the outermost cell layer composed of many specialized types of epidermal cells, such as pavement cells, trichomes, and guard cells. The homeodomain-leucine zipper (HD-ZIP) class Ⅳ transcription factors (TFs), which are unique to the plant kingdom, have been recognized as key regulators of epidermis development. Unlike animal HD proteins, which can bind to DNA as monomers, plant HD-ZIP class Ⅳ TFs bind to DNA as dimers, although little is known about the regulation of their dimerization process. Here, we show that the homodimerization of ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) - HD-ZIP class Ⅳ TF that is required for protoderm development - is regulated by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. We found that ATML1 forms homodimer through interaction via its ZIP motif in yeast and plant cells, although the interaction is abolished by generating a mutation into the lipid-binding START domain to disrupt the lipid-binding ability. These results suggest that lipidic ligands function as key regulators of protoderm development via modulating the dimerization of ATML1.

Multimodal regulation of encystation in Giardia duodenalis revealed by deep proteomics

Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this with published transcriptomic data. We reproducibly identified 3863 (64.5% of Giardia proteins) and quantified 3382 proteins (56.5% of Giardia proteins) over standard trophozoite growth (TY), during low-bile encystation priming (LB), 16 h into encystation (EC), and at cyst maturation (C). This work provides the first known expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways, and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the steroidogenic acute regulatory protein–related lipid transfer (StARkin), oxysterol binding protein related protein (ORP/Osh) and glycosphingolipid transfer protein (GLTP) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts, and found evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first known exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission-blocking anti-giardials.

Targeting Them1 for the Management of Obesity‐Related Disorders

Background Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is a fatty acyl-CoA thioesterase that hydrolyzes long-chain fatty acyl-CoAs into free fatty acids plus CoASH. Genetic disruption of Them1 in mice leads to increased energy expenditure, along with resistance to high fat diet-induced obesity, diabetes and non-alcoholic fatty liver disease. Them1 is comprised of a N-terminal enzymatic domain linked to a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain that can allosterically modulate enzymatic activity. Taken together, these findings suggest that chemical inhibition of Them1 could be leveraged in the management of obesity-related disorders. Aim This study was designed to develop a small molecule inhibitor that targets Them1 activity. Methods Recombinant Them1 protein was expressed in E. coli and purified using a N-terminal His-tag. A fluorescence-based assay to detect free CoASH liberated by the activity of Them1 was optimized and miniaturized into 384-well plates. A high-throughput screen utilizing a highly diverse small molecule library (360,705 compounds) was conducted to identify small molecule inhibitors targeting Them1 activity. The threshold for small molecule inhibitors was set to normalized percent inhibition > 30 and z-score < -3. To select Them1-specific small molecule inhibitors, a counter screen was conducted targeting other Acot isoforms, as well as a truncated Them1 containing only the enzymatic domain (i.e. lacking the START domain). To further improve the potency of small molecules to inhibit Them1 activity, initial structure activity relationship (SAR) expansion was performed. Biomolecular microscale thermophoresis assays were utilized to determine binding affinities of small molecule inhibitors to Them1. Results 330 small molecules were identified from the high-throughput screen (hit rate 0.09%). Ten small molecules derived from the counter screen showed specificity toward inhibiting Them1 activity either through the enzymatic (3 compounds; IC50: 4 – 10 μM) or START (7 compounds; IC50: 6 – 20 μM) domains. SAR analyses revealed molecular features that influenced the IC50 values for selected small molecules. Three small molecules exhibited Kd values in the low μM range. Conclusions We have identified small molecule inhibitors targeting Them1 activity. An optimized small molecule inhibitor targeting Them1 activity should become an attractive candidate for the pharmacologic management of obesity-related disorders.

Single START-domain protein Mtsp17 is involved in transcriptional regulation in Mycobacterium smegmatis.

Tuberculosis caused by the pathogen Mycobacterium tuberculosis (MTB), remains a significant threat to global health. Elucidating the mechanisms of essential MTB genes provides an important theoretical basis for drug exploitation. Gene mtsp17 is essential and is conserved in the Mycobacterium genus. Although Mtsp17 has a structure closely resembling typical steroidogenic acute regulatory protein-related lipid transfer (START) family proteins, its biological function is different. This study characterizes the transcriptomes of Mycobacterium smegmatis to explore the consequences of mtsp17 downregulation on gene expression. Suppression of the mtsp17 gene resulted in significant down-regulation of 3% and upregulation of 1% of all protein-coding genes. Expression of desA1, an essential gene involved in mycolic acid synthesis, and the anti-SigF antagonist MSMEG_0586 were down-regulated in the conditional Mtsp17 knockout mutant and up-regulated in the Mtsp17 over-expression strain. Trends in the changes of 70 of the 79 differentially expressed genes (Log2 fold change > 1.5) in the conditional Mtsp17 knockout strain were the same as in the SigF knockout strain. Our data suggest that Mtsp17 is likely an activator of desA1 and Mtsp17 regulates the SigF regulon by SigF regulatory pathways through the anti-SigF antagonist MSMEG_0586. Our findings indicate the role of Mtsp17 may be in transcriptional regulation, provide new insights into the molecular mechanisms of START family proteins, and uncover a new node in the regulatory network of mycobacteria.

Overexpression of STARD3 attenuates oxidized LDL-induced oxidative stress and inflammation in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is the most common cause of visual disorder in aged people and may lead to complete blindness with ageing. The major clinical feature of AMD is the presence of cholesterol enriched deposits underneath the retinal pigment epithelium (RPE) cells. The deposits can induce oxidative stress and inflammation. It has been suggested that abnormal cholesterol homeostasis contributes to the pathogenesis of AMD. However, the functional role of defective cholesterol homeostasis in AMD remains elusive. STARD proteins are a family of proteins that contain a steroidogenic acute regulatory protein-related lipid transfer domain. There are fifteen STARD proteins in mammals and some, such as STARD3, are responsible for cholesterol trafficking. Previously there was no study of STARD proteins in retinal cholesterol metabolism and trafficking. Here we examined expression of the Stard3 gene in mouse retinal and RPE cells at ages of 2 and 20 months. We found that expression of Stard 3 gene transcripts in both mouse RPE and retina was significantly decreased at age of 20 months when compared to that of age 2 months old. We created a stable ARPE-19 cell line overexpressing STARD3 and found this resulted in increased cholesterol efflux, reduced accumulation of intracellular oxidized LDL, increased antioxidant capacity and lower levels of inflammatory cytokines. The data suggested that STARD3 is a potential target for AMD through promoting the removal of intracellular cholesterol and slowing the disease progression.

Disruption of steroidogenic acute regulatory protein-related lipid transfer domain containing 10 (STARD10) prevents the development of nonalcoholic steatohepatitis (NASH)

Disruption of steroidogenic acute regulatory protein-related lipid transfer domain containing 10 (STARD10) prevents the development of nonalcoholic steatohepatitis (NASH)

A START domain-containing protein is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha

The appropriate regulation of thylakoid lipid synthesis is essential for the function of chloroplasts. In plant cells, membrane lipids synthesized in the ER are utilized as a precursor for the synthesis of chloroplast glycolipids. This pathway is thought to be mediated by the transport of glycerolipids synthesized in the ER into chloroplasts. However, we have little knowledge about the proteins involved in the lipid transfer between these organelles in plant cells. Here we show a protein, STAR2, containing the START (Steroidogenic acute regulatory protein-related lipid transfer) domain known to function as a lipid transporter, is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha. We found that STAR2 localizes on the chloroplast envelope membrane as a punctuate structure and is required for the increase of C20 fatty acids, which are synthesized in the ER, in chloroplast glycolipids in response to phosphate deprivation. Our results indicate that STAR2 of M. polymorpha is likely to be involved in the lipid transfer from ER to chloroplast, presumably as a lipid transporter.

The Them1 START Domain Regulates Nuclear Trafficking in Brown Adipocytes

Thioesterase superfamily member 1 (Them1) is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is a fatty acyl‐CoA thioesterase with tandem hotdog fold thioesterase domains, an N‐terminal set of phosphorylation sites at S15, 18, and 25, and a C‐terminal lipid‐binding steroidogenic acute regulatory protein‐related lipid transfer (START) domain, which modulates thioesterase activity. Cell fractionation studies have demonstrated that Them1 thioesterase activity is localized in microsomes and nuclei, suggesting that it is targeted to specific intracellular compartments. Our aim was to determine the mechanism by which Them1 traffics to the nucleus and its function therein.MethodsIdentification of a putative nuclear localization signal was determined using cNLS mapper ( http://nls‐mapper.iab.keio.ac.jp/cgi‐bin/NLS_Mapper_form.cgi). Full‐length, mutant, or truncated THEM1 constructs were cloned and linked to EGFP. Plasmids or adenoviral vectors were transfected into a differentiated brown adipocyte cell line (MUCAR cells) that does not express Them1. The localization of Them1 was visualized by confocal microscopy via EFGP and mRNA expression was determined using qPCR. Confocal z‐stacks were used to produce 3‐D images with Volocity software. Them1 nuclear localization was evaluated in the presence or absence of the nuclear export inhibitor leptomycin B. Cyclohexamide was used to block new protein synthesis.ResultsPhosphomimic mutations that simulated Them1 phosphorylation at S15 resulted in a diffuse Them1 localization. Stimulation of cells with norepinephrine resulted in Them1 phosphorylation and its reorganization from discrete puncta to a diffuse localization. In the diffuse state, Them1 was transported into the nucleus. Treatment with cyclohexamide suggested that the nuclear Them1 pool was not newly synthesized. We identified a nuclear localization signal in a hinge region located between the 2nd thioesterase domain and the START domain. Deletion of either the nuclear localization signal or the START domain in full length Them 1 blocked nuclear targeting. Conversely, mutant Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus, as evidenced by exit blockade using leptomycin B. Nuclear Them1 regulated mRNA expression levels for a number of genes that regulate lipid metabolism in brown adipocytes.ConclusionsOur results demonstrate that trafficking of Them1 to the nucleus occurs via a nuclear localization signal within the START domain. We speculate that nuclear Them1 regulates the transcription of lipid metabolic enzymes by a mechanism that is distinct from its acyl‐CoA thioesterase activity.Support or Funding InformationThis work was supported by NIH DK103046 to DEC, SJH and EAO.

The START-domain proteins in intracellular lipid transport and beyond

The Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) domain is a ~210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for lipid binding. The helix-grip fold structure defines a large superfamily of proteins, and this review focuses on the mammalian START domain family members that include single START domain proteins with identified ligands, and larger multi-domain proteins that may have novel roles in metabolism. Much of our understanding of the mammalian START domain proteins in lipid transport and changes in metabolism has advanced through studies using knockout mouse models, although for some of these proteins the identity and/or physiological role of ligand binding remains unknown. The findings that helped define START domain lipid-binding specificity, lipid transport, and changes in metabolism are presented to highlight that fundamental questions remain regarding the biological function(s) for START domain-containing proteins.

Abstract 5522: Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity

Abstract The tumor suppressor gene DLC1 encodes a multi-domain protein, including a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, RhoB, and RhoC, and has been hypothesized to be the basis of its tumor suppressor functions. Other well recognized DLC1 domains are an N-terminal sterile alpha motif (SAM) domain, and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. DLC1 is down-regulated in several malignancies by gene deletion, epigenetic promoter methylation, or post-translational mechanisms. In addition, we have recently determined, by analyzing the TCGA database that point mutations in the DLC1 gene occur frequently, and have provided evidence that most of the mutant proteins display attenuated tumor suppressor function. Moreover, the mutations are distributed along the entire coding region, implying that regions of DLC1 in addition to its RhoGAP domain contribute to its full tumor suppressor activity. Several DLC1 binding partners, in addition to Rho-GTP, have been identified. They include Caveolin-1, the principal structural component and marker of caveolae, which interacts with the START domain of DLC1. The distribution of caveolae is closely related to intracellular events, such as MAPK activation or the control of intracellular Ca2+ levels. Phospholipase C delta 1 (PLCD1) transforms PtdIns(4,5)P2 (PIP2) into inositol triphosphate (IP3) and diacyglycerol (DAG), which participate in intracellular Ca2+ mobilization and protein kinase C activation, respectively. p122RhoGAP, the rat homolog of DLC1, was initially cloned as a novel PLCD1-interacting protein, but the interacting region of PLCD1 with p122RhoGAP was not identified. In addition, others have concluded the function of PLCD1 is not affected by human DLC1. In the current study, we have characterized a colon cancer-associated DLC1 mutant (R947C) whose mutation lies in the START domain. The mutant has an attenuated tumor suppressor phenotype, as determined by deficiency in reduction of growth in soft agar and cell migration. Compared with the wild type, the binding of the mutant to PLCD1 and to Caveolin-1 is reduced, but its RhoGAP activity is intact. In addition, phosphorylation of Caveolin-1 by Src at Tyr 14 reduces its interaction with wild type DLC1, and Caveolin-1 and PLCD1 interact in vivo more efficiently with each other in the absence of DLC1. Moreover, wild type DLC1, Caveolin-1 and PLCD1 possess cooperative tumor suppressor activities in cells expressing all three genes. Overall, this study provides new insight into the role of the START domain of DLC1, and reinforces the Rho-GAP-independent regulation of tumor growth by DLC1. Citation Format: Beatriz Sanchez-Solana, Xiaolan Qian, Dunrui Wang, Marian Durkin, Brajendra Tripathi, Alex Papageorge, Douglas R. Lowy. Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5522.

Genome-wide identification, expansion, and evolution analysis of homeobox genes and their expression profiles during root development in carrot.

The homeobox gene family, a large family represented by transcription factors, has been implicated in secondary growth, early embryo patterning, and hormone response pathways in plants. However, reports about the information and evolutionary history of the homeobox gene family in carrot are limited. In the present study, a total of 130 homeobox family genes were identified in the carrot genome. Specific codomain and phylogenetic analyses revealed that the genes were classified into 14 subgroups. Whole genome and proximal duplication participated in the homeobox gene family expansion in carrot. Purifying selection also contributed to the evolution of carrot homeobox genes. In Gene Ontology (GO) analysis, most members of the HD-ZIP III and IV subfamilies were found to have a lipid binding (GO:0008289) term. Most HD-ZIP III and IV genes also harbored a steroidogenic acute regulatory protein-related lipid transfer (START) domain. These results suggested that the HD-ZIP III and IV subfamilies might be related to lipid transfer. Transcriptome and quantitative real-time PCR (RT-qPCR) data indicated that members of the WOX and KNOX subfamilies were likely implicated in carrot root development. Our study provided a useful basis for further studies on the complexity and function of the homeobox gene family in carrot.