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Insights into DEPTOR regulation from in silico analysis of DEPTOR complexes

Teh AH, Yeap KH, Hisano T

J Struct Biol, 2020 11 01;212(2):107602.
PMID: 32798656 DOI: 10.1016/j.jsb.2020.107602

DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the 286SSGYFS291 degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that the two PDZ domains of a DEPTOR dimer bind separately to the dimeric mTOR's FAT domains ~130 Å apart, while each of the two extended linkers is sufficiently long to span from the FAT domain to the kinase domain of mTOR and beyond to join a shared dimer of the DEP domains. This places the linker's S299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards DEPTOR's degron. The CK1α kinase is reportedly responsible for the phosphorylation of the degron, and our docking analysis further reveals that CK1α contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of the degron's S291. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCFβ-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve S293 and S299, may be critical to controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism*
Fulltext Genetic associations of the INSIG2 rs7566605 polymorphism with obesity-related metabolic traits in Malaysian Malays

Apalasamy YD, Moy FM, Rampal S, Bulgiba A, Mohamed Z

Genet. Mol. Res., 2014;13(3):4904-10.
PMID: 25062423 DOI: 10.4238/2014.July.4.4

A genome-wide association study showed that the tagging single nucleotide polymorphism (SNP) rs7566605 in the insulin-induced gene 2 (INSIG2) was associated with obesity. Attempts to replicate this result in different populations have produced inconsistent findings. We aimed to study the association between the rs7566605 SNP with obesity and other metabolic parameters in Malaysian Malays. Anthropometric and obesity-related metabolic parameters and DNA samples were collected. We genotyped the rs7566605 polymorphism in 672 subjects using real-time polymerase chain reaction. No significant associations were found between the rs7566605 tagging SNP of INSIG2 with obesity or other metabolic parameters in the Malaysian Malay population. The INSIG2 rs7566605 SNP may not play a role in the development of obesity-related metabolic traits in Malaysian Malays.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism
Proteomics, oxidative stress and male infertility

Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M

Reprod Biomed Online, 2014 Jul;29(1):32-58.
PMID: 24813754 DOI: 10.1016/j.rbmo.2014.02.013

Oxidative stress has been established as one of the main causes of male infertility and has been implicated in many diseases associated with infertile men. It results from high concentrations of free radicals and suppressed antioxidant potential, which may alter protein expression in seminal plasma and/or spermatozoa. In recent years, proteomic analyses have been performed to characterize the protein profiles of seminal ejaculate from men with different clinical conditions, such as high oxidative stress. The aim of the present review is to summarize current findings on proteomic studies performed in men with high oxidative stress compared with those with physiological concentrations of free radicals, to better understand the aetiology of oxidative stress-induced male infertility. Each of these studies has suggested candidate biomarkers of oxidative stress, among them are DJ-1, PIP, lactotransferrin and peroxiredoxin. Changes in protein concentrations in seminal plasma samples with oxidative stress conditions were related to stress responses and to regulatory pathways, while alterations in sperm proteins were mostly associated to metabolic responses (carbohydrate metabolism) and stress responses. Future studies should include assessment of post-translational modifications in the spermatozoa as well as in seminal plasma proteomes of men diagnosed with idiopathic infertility. Oxidative stress, which occurs due to a state of imbalance between free radicals and antioxidants, has been implicated in most cases of male infertility. Cells that are in a state of oxidative stress are more likely to have altered protein expression. The aim of this review is to better understand the causes of oxidative stress-induced male infertility. To achieve this, we assessed proteomic studies performed on the seminal plasma and spermatozoa of men with high levels of oxidative stress due to various clinical conditions and compared them with men who had physiological concentrations of free radicals. A variety of sperm and seminal plasma proteins were found to be expressed either in abundance (over-expressed) or in a lesser amount (underexpressed), while other proteins were found to be unique either to men with oxidative stress or to men with a balanced ratio of antioxidants/free radicals. Each study included in this review suggested several proteins that could possibly act as biomarkers of oxidative stress-induced male infertility, such as protein DJ-1, PIP, lactotransferrin and peroxiredoxin. Pathway analysis performed in these studies revealed that the changes in seminal plasma proteins in men with oxidative stress could be attributed to stress responses and regulatory pathways, while changes in sperm proteins were linked to stress responses and metabolic responses. Subsequent studies could look into post-translational modifications in the protein profile of men with idiopathic infertility. We hope that the information in this review will contribute to a better understanding of the main causes of idiopathic male infertility.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism
CIP2A expression in high grade prostatic intraepithelial neoplasia and prostate adenocarcinoma: a tissue mıcroarray study

Celikden SG, Baspinar S, Ozturk SA, Karaibrahimoglu A

Malays J Pathol, 2020 Aug;42(2):227-236.
PMID: 32860375

INTRODUCTION: CIP2A is an oncoprotein involved in the progression of several human malignancies. It has recently been described as a prognostic marker in many cancers. The present study aimed to investigate the immunohistochemical expression of CIP2A in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PC), and to analyse the association with the clinicopathological parameters in PC cases to define its role in the development and progression of PC.
MATERIALS AND METHODS: Immunohistochemical staining for CIP2A was performed on the tissue microarray sections of 105 PC, 27 HGPIN and 27 BPH tissues. The CIP2A expression scores were compared with several clinicopathological parameters.
RESULTS: CIP2A was expressed in 96,2% of PC, 55,6% of HGPIN and 40,7% of BPH tissues. The expression of CIP2A in PC was significantly higher than in HGPIN (p<0.0001) and BPH (p<0.0001) cases. CIP2A expression score was significantly associated with Gleason score (p=0.032) and lymphovascular invasion (p=0.039). Nevertheless, there was no statistically significant association between the expression of CIP2A and perineural invasion, pT stage, metastasis and recurrence (p>0.05). Multivariate analysis indicated that GS, lymphovascular invasion, distant metastasis were independent prognostic factors for PC patients but, CIP2A expression score was not found to be a prognostic factor. Additionally, there was no significant difference between the survival times of patients according to CIP2A expression (p=0.174).
CONCLUSIONS: According to our results, the expression of CIP2A protein is increased in PC and its expression may be involved in the development, differentiation, and aggressiveness of PC. However, further studies are needed to confirm our findings and to clarify the role of CIP2A in the development of PC.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism*
A comparative study of the expression of Wnt-1, WISP-1, survivin and cyclin-D1 in colorectal carcinoma

Khor TO, Gul YA, Ithnin H, Seow HF

Int J Colorectal Dis, 2006 May;21(4):291-300.
PMID: 16041507

BACKGROUND AND AIMS: It is well accepted that activation of Wnt signalling occurs in colorectal carcinoma (CRC), but the correlation amongst the various proteins involved in primary tumours are still unclear. The expression of the inducer of this pathway, Wnt-1, and the downstream effectors, WISP-1, cyclin-D1 and survivin proteins, was compared in a series of CRC tissues with the apparently normal adjacent tissues to determine the relationship of these proteins.
PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tissue samples of 47 CRCs surgically resected at the Kuala Lumpur Hospital (KLH) between 1999 and 2000 were used. Immunohistochemical staining with monoclonal antibodies against cyclin-D1 and survivin and polyclonal antibodies against Wnt-1 and WISP-1 was performed. Results of immunohistochemistry were analysed for correlation between biomolecules and histopathological data of the patients.
RESULTS: Of the 47 CRCs, 26 (55.3%), 15 (31.9%), 5 (10.6%) and 28 (59.6%) of the tumours exhibited positivity for Wnt-1, WISP-1, cyclin D1 and survivin, respectively. A lower percentage of the 40 apparently normal adjacent tissues were found to be positive for Wnt-1 (7, 17.5%), WISP-1 (+/-5, 12.5%) and survivin (13, 32.5%), but cyclin D1 was not detected in any of them. Interestingly, the total scores of Wnt-1, WISP-1 and survivin were significantly higher in CRC tissues (p=0.001, 0.034 and 0.044, respectively). Using the Spearman rank correlation test, a positive linear relationship was found between total Wnt-1 score with total WISP-1 score (rho=0.319, p=0.003) and total survivin score (rho=0.609, p=or<0.001). The expression of WISP-1 in the CRC tissues was found to be positively correlated with patients older than 60 years old (p=0.011). In addition, nuclear cyclin-D1 expression was found to be associated with poorly differentiated CRC tissues (p<0.001, Table 5) and right-sided CRC tumour (p=0.019, Table 6). Total WISP-1 score was associated with well-differentiated CRC tissues (p=0.029).
CONCLUSIONS: Overexpression and interplay between Wnt-1, WISP-1, survivin and cyclin-D1 may play a role in tumorigenesis, possibly by promoting cell cycle checkpoint progression, accelerating cell growth and inhibiting apoptosis. Our data may provide useful information towards the search for potent therapeutic targets towards the development of novel treatment strategies for CRC.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism*
The Emerging Role of the Hippo Pathway in Lung Cancers: Clinical Implications

Teoh SL, Das S

Curr Drug Targets, 2017 Nov 30;18(16):1880-1892.
PMID: 27628948 DOI: 10.2174/1389450117666160907153338

BACKGROUND: The incidence of lung cancers has increased globally. Increased exposure to tobacco, passive smoking, less consumption of vegetables and fruits and occupational exposure to asbestos, arsenic and chromium are the main risk factors. The pathophysiology of lung cancer is complex and not well understood. Various microRNAs, genes and pathways are associated with lung cancers. The genes involved in lung cancers produce proteins involved in cell growth, differentiation, different cell cycles, apoptosis, immune modulation, tumor spread and progression. The Hippo pathway (also known as the Salvador-Warts-Hippo pathway) is the latest emerging concept in cancers. The Hippo pathway plays an important role in controlling the size of the tissue and organ by virtue of its action on cell proliferation and apoptosis.
OBJECTIVE: In the present review, we highlight the mammalian Hippo pathway, role of its core members, its upstream regulators, downstream effectors and the resistance cases in lung cancers.
RESULTS: Specific interaction of Mer with cell surface hyaluronan receptor CD44 is vital in cell contact inhibition, thereby activating Hippo pathway. Both transcription co-activators YAP and TAZ (also known as WWTR1, being homologs of Drosophila Yki) are important regulators of proliferation and apoptosis, and serve as major downstream effectors of the Hippo pathway. Mutation of NF2, the upstream regulator of Hippo pathway is linked to the cancers.
CONCLUSION: Targeting YAP and TAZ may be important for future drug delivery and treatment.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism
A Cyclic Peptide Inhibitor of the iNOS-SPSB Protein-Protein Interaction as a Potential Anti-Infective Agent

Sadek MM, Barlow N, Leung EWW, Williams-Noonan BJ, Yap BK, Shariff FM, et al.

ACS Chem. Biol., 2018 10 19;13(10):2930-2938.
PMID: 30226743 DOI: 10.1021/acschembio.8b00561

SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( KD = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism
Fulltext Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth

Oon CE, Bridges E, Sheldon H, Sainson RCA, Jubb A, Turley H, et al.

Oncotarget, 2017 Jun 20;8(25):40115-40131.
PMID: 28445154 DOI: 10.18632/oncotarget.16969

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism*
Cis-9, Trans-11 Conjugated Linoleic Acid Reduces Phosphoenolpyruvate Carboxykinase Expression and Hepatic Glucose Production in HepG2 Cells

Chai BK, Al-Shagga M, Pan Y, Then SM, Ting KN, Loh HS, et al.

Lipids, 2019 06;54(6-7):369-379.
PMID: 31124166 DOI: 10.1002/lipd.12154

Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 μM of c9,t11-CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12-CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11-CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose-6-phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12-CLA increased the expression of G6PC, suggesting the isomer-specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator-activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co-administration of c9,t11-CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12-CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11-CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism
Fulltext Cellular Homeostasis and Antioxidant Response in Epithelial HT29 Cells on Titania Nanotube Arrays Surface

Smn Mydin RB, Sreekantan S, Hazan R, Farid Wajidi MF, Mat I

Oxid Med Cell Longev, 2017;2017:3708048.
PMID: 28337249 DOI: 10.1155/2017/3708048

Cell growth and proliferative activities on titania nanotube arrays (TNA) have raised alerts on genotoxicity risk. Present toxicogenomic approach focused on epithelial HT29 cells with TNA surface. Fledgling cell-TNA interaction has triggered G0/G1 cell cycle arrests and initiates DNA damage surveillance checkpoint, which possibly indicated the cellular stress stimuli. A profound gene regulation was observed to be involved in cellular growth and survival signals such as p53 and AKT expressions. Interestingly, the activation of redox regulator pathways (antioxidant defense) was observed through the cascade interactions of GADD45, MYC, CHECK1, and ATR genes. These mechanisms furnish to protect DNA during cellular division from an oxidative challenge, set in motion with XRRC5 and RAD50 genes for DNA damage and repair activities. The cell fate decision on TNA-nanoenvironment has been reported to possibly regulate proliferative activities via expression of p27 and BCL2 tumor suppressor proteins, cogent with SKP2 and BCL2 oncogenic proteins suppression. Findings suggested that epithelial HT29 cells on the surface of TNA may have a positive regulation via cell-homeostasis mechanisms: a careful circadian orchestration between cell proliferation, survival, and death. This nanomolecular knowledge could be beneficial for advanced medical applications such as in nanomedicine and nanotherapeutics.

Matched MeSH terms: Intracellular Signaling Peptides and Proteins/metabolism

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