Publications by authors named "Ramesh Rijal"

8 Publications

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An Autocrine Negative Feedback Loop Inhibits Dictyostelium discoideum Proliferation through Pathways Including IP3/Ca.

mBio 2021 06 22;12(3):e0134721. Epub 2021 Jun 22.

Department of Biology, Texas A&M University, College Station, Texas, USA.

Little is known about how eukaryotic cells can sense their number or spatial density and stop proliferating when the local density reaches a set value. We previously found that Dictyostelium discoideum accumulates extracellular polyphosphate to inhibit its proliferation, and this requires the G protein-coupled receptor GrlD and the small GTPase RasC. Here, we show that cells lacking the G protein component Gβ, the Ras guanine nucleotide exchange factor GefA, phosphatase and tensin homolog (PTEN), phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) receptor-like protein A (IplA), polyphosphate kinase 1 (Ppk1), or the TOR complex 2 component PiaA have significantly reduced sensitivity to polyphosphate-induced proliferation inhibition. Polyphosphate upregulates IP3, and this requires GrlD, GefA, PTEN, PLC, and PiaA. Polyphosphate also upregulates cytosolic Ca, and this requires GrlD, Gβ, GefA, RasC, PLC, IplA, Ppk1, and PiaA. Together, these data suggest that polyphosphate uses signal transduction pathways including IP3/Ca to inhibit the proliferation of D. discoideum. Many mammalian tissues such as the liver have the remarkable ability to regulate their size and have their cells stop proliferating when the tissue reaches the correct size. One possible mechanism involves the cells secreting a signal that they all sense, and a high level of the signal tells the cells that there are enough of them and to stop proliferating. Although regulating such mechanisms could be useful to regulate tissue size to control cancer or birth defects, little is known about such systems. Here, we use a microbial system to study such a mechanism, and we find that key elements of the mechanism have similarities to human proteins. This then suggests the possibility that we may eventually be able to regulate the proliferation of selected cell types in humans and animals.
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http://dx.doi.org/10.1128/mBio.01347-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262924PMC
June 2021

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells.

Proc Natl Acad Sci U S A 2020 12 2;117(50):31923-31934. Epub 2020 Dec 2.

Department of Biology, Texas A&M University, College Station, TX 77843-3474

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by or human macrophages. In contrast, and accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by or macrophages. Adding extracellular polyphosphate increased survival after phagocytosis by and macrophages. Reducing expression of polyphosphate kinase 1 in reduced extracellular polyphosphate and reduced survival in and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed or cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed and Polyphosphate inhibited phagosome acidification and lysosome activity in and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.
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http://dx.doi.org/10.1073/pnas.2012009117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749317PMC
December 2020

Functional Characterisation of the Autophagy ATG12~5/16 Complex in .

Cells 2020 05 9;9(5). Epub 2020 May 9.

Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. single, double, and triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.
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http://dx.doi.org/10.3390/cells9051179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290328PMC
May 2020

Extracellular signaling in Dictyostelium.

Int J Dev Biol 2019 ;63(8-9-10):395-405

Department of Biology, Texas A∧M University, College Station, Texas, USA.

In the last few decades, we have learned a considerable amount about how eukaryotic cells communicate with each other, and what it is the cells are telling each other. The simplicity of Dictyostelium discoideum, and the wide variety of available tools to study this organism, makes it the equivalent of a hydrogen atom for cell and developmental biology. Studies using Dictyostelium have pioneered a good deal of our understanding of eukaryotic cell communication. In this review, we will present a brief overview of how Dictyostelium cells use extracellular signals to attract each other, repel each other, sense their local cell density, sense whether the nearby cells are starving or stressed, count themselves to organize the formation of structures containing a regulated number of cells, sense the volume they are in, and organize their multicellular development. Although we are probably just beginning to learn what the cells are telling each other, the elucidation of Dictyostelium extracellular signals has already led to the development of possible therapeutics for human diseases.
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http://dx.doi.org/10.1387/ijdb.190259rgDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986813PMC
July 2020

Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in .

Cells 2019 01 19;8(1). Epub 2019 Jan 19.

Institute of Biochemistry I, Medical Faculty, University Hospital Cologne, 50931 Cologne, Germany.

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of in AX2 wild-type and ATG16‾ cells. RNA analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12‾, ATG16‾ and ATG12‾/16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12‾/16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.
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http://dx.doi.org/10.3390/cells8010072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356199PMC
January 2019

An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells.

Mol Biol Cell 2019 01 21;30(2):242-255. Epub 2018 Nov 21.

Department of Biology, Texas A&M University, College Station, TX 77843-3474.

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein-coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.
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http://dx.doi.org/10.1091/mbc.E18-09-0562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589559PMC
January 2019

Expression of N471D strumpellin leads to defects in the endolysosomal system.

Dis Model Mech 2018 09 13;11(9). Epub 2018 Sep 13.

Center for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany

Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Str cells and ectopically expressed Str-GFP or Str-GFP in Str and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to Str At the cellular level, Str cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of Str-GFP in Str cells rescued all observed defects. In contrast, expression of Str-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.033449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177004PMC
September 2018

Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers.

Eur J Cell Biol 2016 Jun-Jul;95(6-7):195-207. Epub 2016 Apr 7.

Centre for Biochemistry, Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany. Electronic address:

p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Our in silico structural models of human and Dictyostelium p97 showed that the disease-causing human R93C, R155H, and R155C as well as Dictyostelium R154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for the Dictyostelium R154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containing Dictyostelium protein as a novel p97 interaction partner. We categorized all UBX-domain containing Dictyostelium proteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses of Dictyostelium UBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human and Dictyostelium UBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease.
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http://dx.doi.org/10.1016/j.ejcb.2016.03.004DOI Listing
February 2017
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