Siddiqui, Shahid (2021) International Worm Meeting "Caenorhabditis elegans Fluorescent Mutants: Tryptophan Kynurenine Pathway"

Siddiqui, Shahid

[International Worm Meeting, 2021]

Caenorhabditis elegans fluorescent mutants (flu-1- flu-4) show altered fluorescence of the lysosome like intestinal granules, when observed under UV light [Babu, 1974, Siddiqui, 1978; Siddiqui and von Ehrenstein, 1980; Siddiqui and Babu, 1980 a, b; and Babu and Bhat, 1980]. Using biochemical techniques, it was shown that the flu-1 mutant alleles characterized by a bluish purple gut fluorescence possess reduced kynurenine hydroxylase enzyme activity [Siddiqui and Babu, 1980], and the flu-2 mutant alleles characterized by a pale dull green fluorescence had a block in kyunreniase enzyme activity [Babu and Bhat, 1980]. The C. elegans genomic sequence encodes these two enzymes in the proximity of flu-1 and flu-2 loci. R07B7.5 encodes kynurenine hydroxylase, and C15H9.7 encodes kynureninase, respectively [Altschul et al., 1990; Kanehisa, 2012]. Kynurenine pathway is the primary route for tryptophan catabolism in and the starting point for the synthesis of nicotinamide adenine dinucleotide (NAD) in mammals. Dysregulation or overactivation of this pathway can result in immune system activation and accumulation of potentially neurotoxic compounds, resulting in inflammation, depression, cancer, neurodegenerative and behavioral alterations, SARS-COV-2 resistance, and epigenetic modifications. Kynurenine pathway genes are also conserved in C. elegans [van der Goot and Nolan, 2013]. We are investigating genetic and epigenetic consequences of manipulating kyunrenine pathway in C. elegans to reveal key components of the catabolic pathway in C. elegans, to reveal molecules and mechanisms that are shared in human disease. For instance, we may use sequence optimized human ortholg cDNA in a flu-1 deletion knockout mutant that shows altered bluish purple intestinal fluorescence and reduced kynurenine hydroxylase activity. Human gene variants cDNA can be used to rescue and be examined for the restoration of intestinal fluorescence and the kynurenine hydroxylase enzyme activity, if the cDNA carrying human variant can restore the normal function in the nematode. Such approach may allow examining human lethal and sublethal variants using the C. elegans model, and could elucidate critical molecular mechanisms underlying human disease.

Siddiqui, Shahid S. et al. (2009) International Worm Meeting "Inhibition of TPA-1 protein kinase C by Enzastaurin: Implications for lung cancer and mesothelioma."

Lognathan, Sivakumar, Salgia, Ravi, Siddiqui, Shahid S.

[International Worm Meeting, 2009]

G-alpha 12/13 types of G-proteins play an important role in development, and are known to function as potent oncogenes when expressed in cultured cells. We have previously identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in C. elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF (Yau et al., 2003). Ronald Plasterk and colleagues used suppressor screen for gpa-12 and identified suppressors in the tpa-1 gene that encodes two protein kinase C isoforms (TPA-1A and TPA-1B) that are homologous to PKC theta/delta (Tabuse et al. 1995; van der Linden et al., 2003). TPA-1 mediates the action of tumor promoter PMA and results in feeding defects and developmental arrest. In human cancers including lung cancer and mesothelioma, PKC function is closely associated with vascular endothelial growth factor receptor (VEGFR). The VEGF (vascular endothelial growth factor) and its receptor VEGFR are up-regulated in a variety of malignant tissues, including lung cancer and mesothelioma. Our results on malignant cell lines (H513, H28, H2461, H2373, H2596, and MSTO) show that PKC and VEGFR are overexpressed in lung and mesothelioma and also in tumor tissues. Inhibition of PKC by Enzastaurin inhibits the phosphorylation of PKC-beta 1 and 2 induced by rhVEGF, and Enzastaurin is under clinical trials for lung cancer. Expression of a reporter GFP under the tpa-1 promoter in C. elegans transgenic lines show expression of the reporter in nervous system sensory neurons, muscle cells, and somatic vulva, and this expression is reduced in animals exposed to Enzastaurin. This system provide high throughput screening platform for evaluating potential cancer drugs that mediate conserved signal transduction pathways across C. elegans and humans, and reveal novel genes that interact with PKC, VEGF and G-protein signaling. We thank J. Miwa and Y. Tabuse for discussion and strains.

Siddiqui, Shahid S. et al. (2013) International Worm Meeting "Antimicrobial compound screens using C. elegans model system for periodontal pathogens -."

Siddiqui, Shahid S., Al-Beyari, Mohammad, Faskhani, Fathy A.

[International Worm Meeting, 2013]

Chronic periodontal disease (PD) is an inflammatory condition that is highly widespread. Periodontal disease is a result of sub-gingival plaque pathogens, leading to alveolar bone loss. PD is also associated with diabetes, obesity, cardiovascular disease, respiratory disease, rheumatic arthritis and pre-term birth. The mechanism of periodontal infection is poorly understood due to the complexity of microbial factors, and highly complex inflammatory and anti-inflammatory signaling between pathogens and the host. New microbial pathogens are emerging that are resistant to conventional antibiotics; thus there is a need for drugs that are new class of antibiotics, and novel compounds that reduce pathogenicity of invading microbes. Pioneering studies in Frederick Ausubel's Laboratory (Harvard Medical School) have shown the effective use of C. elegans as a simple and powerful genetic model to screen for antimicrobial compounds, such as the discovery of RPW-24 that protects C. elegans from bacterial infections by modulating the p38 MAPK, and ATF7 transcription regulatory pathways [Pukkila-Worley et al. 2012]. We have previously used C. elegans to screen chemicals by using the nematode egg laying assay, and have identified novel compounds that affect egg laying behavior in a high throughput screen of commercially available chemical library [Siddiqui et al., C. elegans meeting UCLA, 2011]. We are screening chemical libraries in C. elegans for novel compounds that may inhibit the growth of oral pathogens such as Pseudomonas aeruginosa, found in the periodontal infections; and look for modulators of NSY-1, SEK-1, PMK-1 Mitogen Activated Protein (MAP) Kinase pathway that is comparable to the mammalian ASK-1(MAP kinase kinase kinase)/MKK3/6 (MAP kinase kinase) p38 MAP kinase signaling pathway. To validate the discovery of novel compounds in C. elegans high throughput screens for periodontal pathogens, we will use in vitro PLC (periodontal ligament cell) cell culture screens. Results will be presented on the efficacy of screened compounds on oral pathogens specific to chronic periodontal disease.

Siddiqui, Shahid S. et al. (2011) International Worm Meeting "Fat Burning in Sleeping Worms: Regulation of Multiple Signaling Pathways by Kruppel Like Factors."

Gozal, David, Yang, Chen, Zhang, Jun, Siddiqui, Shahid S., Dhansingh, Immanuel, Hashmi, Sarwar

[International Worm Meeting, 2011]

Sleep is an important behavior across diverse species, regulated by conserved signaling pathways. OSAS (obstructive sleep apnea syndrome) is characterized by episodic hypoxia, affecting about 5% of adult American population and may lead to hypertension, if left untreated. In C. elegans post-embryonic development is accompanied by four larval stages [Sulston et al., 1983] that are interrupted by periods of inactivity called lethargus, and has been suggested to represent 'sleep' [Raizen et al., 2008]. The cAMP-CREB pathway has been shown to affect sleep-wake cycle in Drosophila, C. elegans and mammalian systems [Zimmerman et al., 2008], and cGMP dependent protein kinase expression is regulated by Rho and KLF4 [Zeng et al., 2006]. Genetic analysis has suggested the role of LIN-3 and LET-23 (the EGF like-ligand and EGF receptor, respectively) in sleep like behavior in C. elegans [Van Buskirk and Sternberg, 2007]. Overexpression of LIN-3 results in quiescent behavior during periods of normal activity, through the activity of ALA neuron that is regulated by paired (a family of Kruppel like transcription factors) and LIM class of transcription factors CEH17, CEH-14/Chx10, and CEH17/Phox2 through a feedback regulatory pathway [Van Buskirk and Sternberg, 2010]. We have shown the role of KLF-3 in beta oxidation of fatty acids in C. elegans that results in accumulation of fat in intestinal cells in klf-3(ok1975) homozygous loss of function mutants, arrested development and poor fecundity [Hashmi et al., 2008; Zhang et al. 2011 submitted], and may also affect sleep cycle by affecting the lethargus timings during larval molts. Interestingly, KLF4 in mammals not only mediates adipogenesis but also regulates kallistatin expression [Shen et al., 2009] that regulates kidney protein expression during episodic hypoxia (EH), by inhibiting vasodilation mediated by kallikrein-Kallistatin signaling pathway [Thonhboonkered et al., 2002]. The sleep regulating egl-4 gain of function mutation in cyclic GMP also results in the accumulation of fat in the intestine [Raizen et al., 2006]. Thus accumulating results suggest that klf-3 signaling pathway analysis in C. elegans may elucidate fat utilization (see the abstract by Hashmi et al., this meeting), sleep, and reproductive behavior.

Shahid Siddiqui et al. (2007) International Worm Meeting "ACTIN BASED TRAFFICKING OF THE ATYPICAL KINESIN VAB-8."

Shahid Siddiqui, Yusuf Ali

[International Worm Meeting, 2007]

The C. elegans genome encodes 20 members of the kinesin family (klp-1 to klp-20) including the conventional kinesin (UNC-116), and 19 other genes encoding kinesin like proteins (Siddiqui 2002). However, the vab-8 is different as it encodes a short and long form of kinesin like proteins VAB8-L and VAB-8S (Wolf et al. 1998); the short VAB-8S lacks the conserved motor domain, and instead contains a myosin like domain. Recently VAB-8, UNC-73 and MIG-2 were shown to control cell migration and cell polarity mediated through UNC-40 (Levy-Strumpf & Culotti, 2007), and further VAB-8 and UNC-73 have been shown to regulate SAX-3 receptor to direct growth cone migration (Goshima et al., 2007). Herein we report that the vab-8 reporter gene is expressed in muscle cells and VAB-8S may utilize actin filaments for its trafficking in cell migration and cellular polarity. By reciprocal immuno-precipitation and immunocytochemical staining we have shown that VAB-8 is associated with actin filaments. The VAB-8S is expressed in the muscle cells and this expression is highly polarized as it is localized in the anterior region of each muscle cell (Wormbase). In contrast the VAB8L is restricted to neural expression and is not expressed in muscle cells. The double mutants of various vab-8 alleles in combination with mutations in actin gene act-123 result in severe paralysis and lethality. To test the genetic interaction of vab-8 with the unc-116 gene we have examined the double mutants of vab-8 mutant alleles e1017, gm84 and ev411, with the unc-116(e2310) and unc-116(rh24) alleles and found that these two kinesins interact physiologically, resulting in phenotypes ranging from defects in locomotion, paralysis and lethality. Expression of the unc-116::lacZ reporter gene in different vab-8 mutant background shows that unc-116 expression depends on the vab-8 gene expression in the posterior half of the animals. We propose a model suggesting that the VAB-8 kinesin may use actin filaments for trafficking and the expression of unc-116 in muscle cells may require the vab-8 gene expression in the posterior half of the animal. Using genetic, biochemical and biophysical methods we will examine the intriguing possibility that kinesin motors may use both microtubules and the actin filaments for the intracellular transport in regulating anterior/posterior polarity and axonal growth. We thank Gian Garriga for the gift of vab-8 mutant alleles and the anti-VAB-8 polyclonal antibodies, and David Baillie for reporter strains. Research supported by NIH and Monbusho grants to SSS.

Shahid S Siddiqui (2003) International Worm Meeting "Expression of the angiomotin like gene in C. elegans"

Shahid S Siddiqui

[International Worm Meeting, 2003]

Kinesin, myosin and dynein are motor proteins that move intracellular cargo in eukaryotic cells. We have earlier identified a family of 20 genes encoding kinesin protein family members in C. elegans (Siddiqui, 2002). Myosin and kinesin motors are characterized by a globular motor domain and a variable tail domain that is mostly non globular and exists in coiled alpha helical structure. Recently a novel angiostatin interacting protein called angiomotin has been identified by its ability to bind angiostatin in a yeast dihybrid screen (Troyanovsky et al., 2001, Bratt et al., 2002). In C. elegans a homolog of the angiomotin encoded by the F59C12.3 gene has been reported (Bratt et al 2002. We have studied the expression of the angiomotin gene in C. elegans. The nematode angiomotin encodes a single copy gene that has significant homology with the coiled coiled structure of the kinesin (e.g. genes encoded by klp-3, klp-4, klp-8, klp-10, klp-12, klp-18, and klp-19) and myosin (e.g. myo-3) rod regions. Since coiled coiled domains are involved in protein- protein interactions the nematode angiomotin may also function in intracellular transport. Human angiomotin gene AMOT1 is highly expressed in endothelial cells and can inhibit both cell proliferation and cell motility (Troyanovsky et al., 2001). The C. elegans angiomotin gene encodes a 2 kb transcript that is highly expressed both during embryonic (from oocyte stage to the hatching) and postembryonic development (during larval and adult development). The RNAi phenotype using the dsRNA shows a reduction in brood size and reduced oogenesis, but has no apparent morphological abnormalities. The nematode angiomotin is about 23% identical (94/403) and 46% residues show positive identity with the human angiomotin protein. The nematode angiomotin is highly rich in glutamine residues in the amino terminus like mammalian angiomotin, but it lacks the PDZ domain that is highly conserved in mammalian angiomotins located in the extreme carboxyl terminal region of the protein. Since angiomotin is a novel inhibitor of the angiostatin, we are analyzing the interacting genes with the gene encoding angiomotin in C. elegans using forward and reverse genetic analyses.

Shahid S Siddiqui et al. (2003) International Worm Meeting "Regulation of endocytosis in intestinal epithelium by components of TGF-beta Signaling"

Shahid S Siddiqui, Zeba K Siddiqui, Garth Patterson, Asrar B Malik

[International Worm Meeting, 2003]

Previously, in Caenorhabditis elegans a conserved TGF-beta signaling pathway has been characterized (Riddle, 1997, Thomas, 2001). This pathway is known to control dauer formation behavior, similar to the daf-2 insulin like signaling pathway. The daf-7 encodes a homolog of the GDF-8 and is expressed in the chemosensory neuron ASI, and the type I and type II TGF beta Ser/Thr receptor kinases are encoded by daf-1 and daf-4. The downstream Smad proteins are encoded by daf-8, and daf-14 genes that couple the TGF beta signals from DAF-1 and DAF-4 receptor kinases to regulate transcription of downstream genes. We have examined the role of various components of the TGF beta pathway in albumin endocytosis in the intestinal epithelial cells in post-emb ryonic development, and described that mutations in daf-1 and daf-4 inhibit albumin endocytosis in the intestinal cells. Genetic analysis reveals that mutations in daf-3 null mutation completely suppress the endocytosis defective phenotype of daf-1, daf-4 , daf-7 and daf-8 mutations, suggesting an independent endocytosis pathway exposed by mutations in daf-3. The daf-3 encoded protein is homologous to vertebrate DPC4, a cofactor of Smad1, Smad2, and Smad3. In contrast, downstream components of the TGF-beta signaling that affect body size, such as sma-2, sma-3, and sma-6 apparently have no effect on albumin endocytosis in the intestine. We thank D. Riddle, G. Ruvkun, C. Savage, J. Thomas and T. Stiernagle, CGC for strains and L. Jacobson for making the original observation that mutants in daf-4 are defective in albumin uptake.

Shahid S Siddiqui et al. (2001) International C. elegans Meeting "A novel role of the TGF-beta signaling cascade in endocytosis in C. elegans"

Zeba K Siddiqui, Shahid S Siddiqui

[International C. elegans Meeting, 2001]

TGF-beta and related members of the cytokines play a central role in growth and development in higher organisms. In C. elegans three important aspects of development have been described that are mediated through TGF-beta signaling pathway. These include the dauer formation (Estevez et al., 1993), control of body size, and male tail morphogensis (Morita et al., 1999; Suzuki et al, 1999), and the axonal guidance (Clovita et al., 1998). We have found that the TGF-beta signaling also mediates endocytosis in the intestinal epithelium of C. elegans . Mutants in TGF-beta receptor family of Ser/Thr kinases, daf-1 and daf-4 (Georgi et al., 1990; Estevez et al., 1993), show abnormal endocytosis. Activation of TGF-beta receptor induced endocytosis of albumin in intestine is dependent on the endocytic machinery that includes, dynamin, clathrin heavy chain, rab-5, rab-11, and alpha and beta adaptin. We have extended these findings to show the role of TGF-beta receptors in albumin endocytosis in monolayers of endothelial cells by inhibiting endocytosis with receptor antibodies and its augmentation in cells transfected with cDNA encoding the receptor (Attisano et al., 1993). TGF-beta receptor mediated albumin endocytosis specifically activates Smad2 phosphorylation, Smad4 translocation, MAPK p38 phosphorylation, and promotes cell growth and is anti-apoptotic. We propose a model that suggests that activation of endocytosis promotes cell growth and is anti-apoptotic. Thus, interaction of albumin with TGF-beta receptors is critical in regulating its endocytosis and the downstream activation of the TGF-beta signaling pathway. We thank A. Malik for enthusiastic support, and lively discussions, D. Riddle, S. Uddin, L. Jacobson, J. Massague, P. ten-Dijke, K. Miyazono, A. Shajahan, P. Okemma, T. Orenic, T. Stiernagle, and the CGC for strains, reagents and invaluable suggestions.

Shahid S Siddiqui et al. (2005) International Worm Meeting "KLP-4 a Kinesin-3 family member is required for embryogenesis and neural development in C. elegans"

Douglas Yau, Toshio Hanada, Athar Chishti, David Baillie, Asrar B Malik, Shahid S Siddiqui, Hae K Lee, Robert Johnsen, Zhongying Zhao

[International Worm Meeting, 2005]

Members of the UNC-104/KIF-1 family play important role in neural growth, axonal transport and synaptic function. In C. elegans in addition to UNC-104, two similar motors KLP-4 and KLP-6 comprise the Kinesin-3 family subgroup (Siddiqui 2002) characterized by an amino-terminal Motor head domain (ATP and microtubule binding sites), followed by a coiled coil stalk hinge region for multimer formation and a carboxyl terminal tail domain of variable length and composition and is similar to the GAKIN 13A family of mammalian motors (Hanada et al., 2002). We have shown that inhibition of klp-4 function by RNAi an rrf-3 mutant background results in embryonic arrest and reduction in brood size. A klp-4::gfp promoter fusion transgene expresses in the sensory neurons in the head, a set of ventral cord motor neurons, and the sensory ray neurons in the male tail ray neurons and the intestine from larval stages L1-L4 and in adults. Polyclonal antibodies raised against the carboxyl terminal of the KLP-4 detect a 180-kDa band. Immunostaining with an affinity purified anti-KLP-4 antibody shows that the KLP-4 motor is present in the sensory neurons in the head, developing embryos, germ cells and intestine, consistent with the klp-4::gfp expression results. The KLP-4 also contains the fork-head association domain (residues 534-594) suggesting a motif for protein-protein interaction. We are examining the stalk and tail region of the KLP-4 motor using klp-4::gfp constructs to determine the cell specificity of klp-4 expression. Expression of klp-4 closely resembles that of klp-6 another member of the UNC-104 family of kinesins enriched in sensory neurons and motor neurons. A mutation in klp-6 aka lov-2(sy511) has been described (Paden and Barr, 2004). Expression of klp-4::gfp in lov-2 background may reveal epistasis. Similar interactions between klp-6 and unc-104 are under investigation.

Shahid S Siddiqui et al. (2004) West Coast Worm Meeting "Expression of kinesin motor proteins during embryogenesis and neural function in C. elegans"

Robert Jhonsen, Shahid S Siddiqui, M Yusuf Ali, M L Khan, David Baillie, Shams T Khan, Asrar B Malik, Zeba K Siddiqui, Zhongying Zhao, Douglas Yau, Fumio Hori

[West Coast Worm Meeting, 2004]

Kinesin is an ATP hydrolyzing enzyme that transduces chemical energy of ATP hydrolysis into mechanical force to carry intracellular cargo on microtubule tracks. Why do multiple kinesin motors exist in a given cell type or a tissue, and whether these motors perform distinct or overlapping functions, remains to be understood. In C. elegans a superfamily of 21 kinesin motor proteins has been described that participate in a variety of cellular function such as chromosomal movement, cell and growth cone migration, process outgrowth and axonal guidance (Siddiqui, 2002). Members of the nematode kinesin family represent all major groups of kinesin motors found in the vertebrates. Accordingly, we have determined the temporal and spatial pattern of expression of the nematode kinesin family members by in situ hybridization, or by studying kinesin promoter fusion with GFP or dsRed protein markers in transgenic animals. Our results suggest that members of the kinesin family (klp-1-klp-20) have distinct role in mitosis, chromosomal dynamics and are essential for neural development and function.