Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Ce... more Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, e.g. drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. Yet, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograph models in vivo, and also following the fates of individual clones within each...
Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cel... more Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones ...
ABSTRACTThe COVID-19 pandemic has given rise to diverse approaches to track infections. The causa... more ABSTRACTThe COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2, is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy for COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the method’s theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). MPAD demonstrated facile detection of SARS-CoV-2 proteins compared with SARS-CoV-2 RNA via qRT-PCR in wastewater. In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These n...
Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adapt... more Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70–Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also contro...
In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track co... more In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care units, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through measuring trends of viral RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 genes are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada’s national capital region, i.e., the City of Ottawa, ON (pop. ≈ 1.1M) and the City of Gatineau, QC (pop. ≈ 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentra...
Prevention, Early Detection, and Interception, 2019
Pancreatic cancer is one of the deadliest types of cancer, killing over 50,000 each year in the U... more Pancreatic cancer is one of the deadliest types of cancer, killing over 50,000 each year in the US, and with a five-year survival rate below 8%. However, currently there are no reliable diagnostic tests for pancreatic cancer, and the great majority of cases are detected at a late stage, with bleak mortality rates as a result. The ActivSignal IPAD platform monitors activity of 26 cancer signaling pathways in multiplex, by examining phosphorylation status or expression of 70 protein targets. The technology utilizes pairs of antibodies per each protein target to ensure high specificity. These antibody pairs are carefully tested and selected. Detection will only take place if both antibodies in a pair bind to their specific target molecule during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. The IPAD assay requires a very small amount of sample. All these characteristics make it potentially adaptable for diagnostics, simultaneously profiling multiple proteins targets. We have tested the assay in a preliminary analysis of human pancreatic cancer patients. Several control and pancreatic ductal adenocarcinoma (PDAC) blood samples were used for analysis. The assay can be performed on a very small volume of material, and 100 microliters of each sample was obtained from a collaborating academic medical center. The profiling was performed on 40 previously identified, relevant proteins. The comparison of Normal controls and PDAC patient samples gave a clear pattern that discriminated these two population groups with a great degree of certainty. Thus, giving a proof of concept that the assay can be used for diagnostic development for pancreatic cancer detection, an urgent unmet need. Citation Format: Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. Multiplex analysis of proteins for cancer diagnosis: A case study using ActivSignal technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1621.
The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in mu... more The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in multiplex by examining phosphorylation status or expression of 70 protein targets from canonical signaling pathways. The technology uses antibody pairs for each protein target to ensure high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. Human and mouse samples can be analyzed. The IPAD assay requires only a very small amount of your precious sample. Applications include: - Direct analysis of protein activity of the signaling cascades and not subsequent downstream events. - Testing physiological effects of compounds of interest. – Influence on specific protein targets, as well as the signaling pathways as a whole. – Secondary and/or off-target effects. - Similarly, analysis of signaling pathways activation status in response to different stimuli, such as: CRISPR, shRNA, hormones, stresses and other treatments. - Medium-throughput analysis (96-well format). - Tissue sample profiling from patients and animals (murine). - Profiling of tumor biopsies. Our collaborators analyzed the mechanism of action of JG-98, an Hsp70 inhibitor that exhibits anti-cancer activities that affect both cancer cells and tumor associated macrophages. Using gene expression and ActivSignal protein profiling assay, the signaling pathways influenced by JG-98 were identified, which predicted potential drug combinations that can be used with JG-98 for further preclinical development. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc. Citation Format: Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. ActivSignal technology for multiplex analysis of signaling protein activation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2162.
The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling path... more The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways by examining phosphorylation events or expression of more than 60 most relevant human and mouse protein targets. The technology uses antibodies against established and well known targets that reflect activities of canonical pathways. We utilize paired antibodies for each protein target for high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The IPAD assay can be used to profile samples made from cell culture, tissues, blood, etc. The IPAD assay requires only minute amounts of sample, which can be easily taken from your precious sample where available amounts may be limited. Applications include: Direct snapshot of signaling pathway activity rather than downstream effects. Understanding the physiological effects of a ...
Proceedings of the National Academy of Sciences of the United States of America, Jul 24, 2018
Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive respo... more Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 sign...
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer ac... more Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer activities affecting both cancer cells and tumor-associated macrophages. They disrupt Hsp70 interaction with a co-chaperone Bag3 and affect signaling pathways important for cancer development. Due to a prior report that depletion of Hsp70 causes similar responses as depletion of Hsp90, interest to Hsp70 inhibitors as drug prototypes is hampered by potential similarity of their effects to effects of Hsp90 inhibitors. Here, using the Connectivity Map platform we demonstrate that physiological effects of JG-98 are dissimilar from effects of Hsp90 inhibitors, thus justifying development of these compounds. Using gene expression and ActivSignal IPAD platform, we identified pathways modulated by JG-98. Some of these pathways were affected by JG-98 in Bag3-dependent (e.g. ERK) and some in Bag3-independent manner (e.g. Akt or c-myc), indicating multiple effects of Hsp70 inhibition. Further, we ide...
The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that re... more The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knocko...
Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer ... more Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.
Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly un... more Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly understood mechanism. In this study, we demonstrate that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) is a novel tumor suppressor that is highly expressed in normal renal tubular epithelial cells, but it is downregulated in human renal cancer. We have identified CCAAT/enhancer-binding proteinβ (C/EBPβ, also called LAP) as a key transcriptional regulator of TMIGD1, whose loss of expression is responsible for downregulation of TMIGD1 in RCC. Transcriptionally active C/EBPβ/LAP physically interacted with and increased TMIGD1 promoter activity and expression of TMIGD1. Re-introduction of TMIGD1 into renal tumor cells significantly inhibited tumor growth and metastatic behaviors such as morphogenic branching and cell migration. Restoring TMIGD1 expression in renal tumor cells stimulated phosphorylation of p38MAK, induced expression of p21CIP1 (cyclin-dependent kinase inhibitor ...
Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers.... more Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI(+)] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI(+)] and stability of its inheritance. Besides [PSI(+)], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI(+)] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion am...
Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Ce... more Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, e.g. drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. Yet, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograph models in vivo, and also following the fates of individual clones within each...
Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cel... more Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones ...
ABSTRACTThe COVID-19 pandemic has given rise to diverse approaches to track infections. The causa... more ABSTRACTThe COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2, is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy for COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the method’s theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). MPAD demonstrated facile detection of SARS-CoV-2 proteins compared with SARS-CoV-2 RNA via qRT-PCR in wastewater. In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These n...
Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adapt... more Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70–Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also contro...
In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track co... more In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care units, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through measuring trends of viral RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 genes are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada’s national capital region, i.e., the City of Ottawa, ON (pop. ≈ 1.1M) and the City of Gatineau, QC (pop. ≈ 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentra...
Prevention, Early Detection, and Interception, 2019
Pancreatic cancer is one of the deadliest types of cancer, killing over 50,000 each year in the U... more Pancreatic cancer is one of the deadliest types of cancer, killing over 50,000 each year in the US, and with a five-year survival rate below 8%. However, currently there are no reliable diagnostic tests for pancreatic cancer, and the great majority of cases are detected at a late stage, with bleak mortality rates as a result. The ActivSignal IPAD platform monitors activity of 26 cancer signaling pathways in multiplex, by examining phosphorylation status or expression of 70 protein targets. The technology utilizes pairs of antibodies per each protein target to ensure high specificity. These antibody pairs are carefully tested and selected. Detection will only take place if both antibodies in a pair bind to their specific target molecule during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. The IPAD assay requires a very small amount of sample. All these characteristics make it potentially adaptable for diagnostics, simultaneously profiling multiple proteins targets. We have tested the assay in a preliminary analysis of human pancreatic cancer patients. Several control and pancreatic ductal adenocarcinoma (PDAC) blood samples were used for analysis. The assay can be performed on a very small volume of material, and 100 microliters of each sample was obtained from a collaborating academic medical center. The profiling was performed on 40 previously identified, relevant proteins. The comparison of Normal controls and PDAC patient samples gave a clear pattern that discriminated these two population groups with a great degree of certainty. Thus, giving a proof of concept that the assay can be used for diagnostic development for pancreatic cancer detection, an urgent unmet need. Citation Format: Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. Multiplex analysis of proteins for cancer diagnosis: A case study using ActivSignal technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1621.
The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in mu... more The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in multiplex by examining phosphorylation status or expression of 70 protein targets from canonical signaling pathways. The technology uses antibody pairs for each protein target to ensure high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. Human and mouse samples can be analyzed. The IPAD assay requires only a very small amount of your precious sample. Applications include: - Direct analysis of protein activity of the signaling cascades and not subsequent downstream events. - Testing physiological effects of compounds of interest. – Influence on specific protein targets, as well as the signaling pathways as a whole. – Secondary and/or off-target effects. - Similarly, analysis of signaling pathways activation status in response to different stimuli, such as: CRISPR, shRNA, hormones, stresses and other treatments. - Medium-throughput analysis (96-well format). - Tissue sample profiling from patients and animals (murine). - Profiling of tumor biopsies. Our collaborators analyzed the mechanism of action of JG-98, an Hsp70 inhibitor that exhibits anti-cancer activities that affect both cancer cells and tumor associated macrophages. Using gene expression and ActivSignal protein profiling assay, the signaling pathways influenced by JG-98 were identified, which predicted potential drug combinations that can be used with JG-98 for further preclinical development. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc. Citation Format: Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. ActivSignal technology for multiplex analysis of signaling protein activation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2162.
The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling path... more The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways by examining phosphorylation events or expression of more than 60 most relevant human and mouse protein targets. The technology uses antibodies against established and well known targets that reflect activities of canonical pathways. We utilize paired antibodies for each protein target for high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The IPAD assay can be used to profile samples made from cell culture, tissues, blood, etc. The IPAD assay requires only minute amounts of sample, which can be easily taken from your precious sample where available amounts may be limited. Applications include: Direct snapshot of signaling pathway activity rather than downstream effects. Understanding the physiological effects of a ...
Proceedings of the National Academy of Sciences of the United States of America, Jul 24, 2018
Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive respo... more Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 sign...
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer ac... more Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer activities affecting both cancer cells and tumor-associated macrophages. They disrupt Hsp70 interaction with a co-chaperone Bag3 and affect signaling pathways important for cancer development. Due to a prior report that depletion of Hsp70 causes similar responses as depletion of Hsp90, interest to Hsp70 inhibitors as drug prototypes is hampered by potential similarity of their effects to effects of Hsp90 inhibitors. Here, using the Connectivity Map platform we demonstrate that physiological effects of JG-98 are dissimilar from effects of Hsp90 inhibitors, thus justifying development of these compounds. Using gene expression and ActivSignal IPAD platform, we identified pathways modulated by JG-98. Some of these pathways were affected by JG-98 in Bag3-dependent (e.g. ERK) and some in Bag3-independent manner (e.g. Akt or c-myc), indicating multiple effects of Hsp70 inhibition. Further, we ide...
The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that re... more The cytoplasmic-element-binding (CPEB) protein is a sequence-specific RNA-binding protein that regulates cytoplasmic polyadenylation-induced translation. In mouse embryo fibroblasts (MEFs) lacking CPEB, many mRNAs encoding proteins involved in inflammation are misregulated. Correlated with this aberrant translation in MEFs, a macrophage cell line depleted of CPEB and treated with lipopolysaccharide (LPS) to stimulate the inflammatory immune response expresses high levels of interleukin-6 (IL-6), which is due to prolonged nuclear retention of NF-κB. Two proteins involved in NF-κB nuclear localization and IL-6 expression, IκBα and transforming growth factor beta-activated kinase 1 (TAK1), are present at excessively low and high steady-state levels, respectively, in LPS-treated CPEB-depleted macrophages. However, only TAK1 has an altered synthesis rate that is CPEB dependent and CPEB/TAK1 double depletion alleviates high IL-6 production. Peritoneal macrophages isolated from CPEB knocko...
Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer ... more Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.
Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly un... more Renal cell carcinoma (RCC) is a high-risk metastasizing tumor with a poor prognosis and poorly understood mechanism. In this study, we demonstrate that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) is a novel tumor suppressor that is highly expressed in normal renal tubular epithelial cells, but it is downregulated in human renal cancer. We have identified CCAAT/enhancer-binding proteinβ (C/EBPβ, also called LAP) as a key transcriptional regulator of TMIGD1, whose loss of expression is responsible for downregulation of TMIGD1 in RCC. Transcriptionally active C/EBPβ/LAP physically interacted with and increased TMIGD1 promoter activity and expression of TMIGD1. Re-introduction of TMIGD1 into renal tumor cells significantly inhibited tumor growth and metastatic behaviors such as morphogenic branching and cell migration. Restoring TMIGD1 expression in renal tumor cells stimulated phosphorylation of p38MAK, induced expression of p21CIP1 (cyclin-dependent kinase inhibitor ...
Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers.... more Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI(+)] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI(+)] and stability of its inheritance. Besides [PSI(+)], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI(+)] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion am...
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Papers by Ilya Alexandrov