Journal articles: 'HAP1'

1

Rong, Juan, Shihua Li, Guoqing Sheng, Meng Wu, Brian Coblitz, Min Li, Haian Fu, and Xiao-Jiang Li. "14-3-3 Protein Interacts with Huntingtin-associated Protein 1 and Regulates Its Trafficking." Journal of Biological Chemistry 282, no. 7 (December 13, 2006): 4748–56. http://dx.doi.org/10.1074/jbc.m609057200.

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HAP1 (Huntingtin-associated protein 1) consists of two alternately spliced isoforms (HAP1A and HAP1B, which have unique C-terminal sequences) and participates in intracellular trafficking. The C terminus of HAP1A is phosphorylated, and this phosphorylation was found to decrease the association of HAP1A with kinesin light chain, a protein involved in anterograde transport in cells. It remains unclear how this phosphorylation functions to regulate the association of HAP1 with trafficking proteins. Using the yeast two-hybrid system, we found that HAP1 also interacts with 14-3-3 proteins, which are involved in the assembly of protein complexes and the regulation of protein trafficking. The interaction of HAP1 with 14-3-3 is confirmed by their immunoprecipitation and colocalization in mouse brain. Moreover, this interaction is specific to HAP1A and is increased by the phosphorylation of the C terminus of HAP1A. We also found that expression of 14-3-3 decreases the association of HAP1A with kinesin light chain. As a result, there is less HAP1A distributed in neurite tips of PC12 cells that overexpress 14-3-3. Also, overexpression of 14-3-3 reduces the effect of HAP1A in promoting neurite outgrowth of PC12 cells. We propose that the phosphorylation-dependent interaction of HAP1A with 14-3-3 regulates HAP1 function by influencing its association with kinesin light chain and trafficking in neuronal processes.

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Schneider, J. C., and L. Guarente. "Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4." Molecular and Cellular Biology 11, no. 10 (October 1991): 4934–42. http://dx.doi.org/10.1128/mcb.11.10.4934.

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Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.

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Schneider, J. C., and L. Guarente. "Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4." Molecular and Cellular Biology 11, no. 10 (October 1991): 4934–42. http://dx.doi.org/10.1128/mcb.11.10.4934-4942.1991.

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Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.

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Yang, Miao, Yoon Lim, Xiaojiang Li, Jin-Hua Zhong, and Xin-Fu Zhou. "Precursor of Brain-derived Neurotrophic Factor (proBDNF) Forms a Complex with Huntingtin-associated Protein-1 (HAP1) and Sortilin That Modulates proBDNF Trafficking, Degradation, and Processing." Journal of Biological Chemistry 286, no. 18 (February 28, 2011): 16272–84. http://dx.doi.org/10.1074/jbc.m110.195347.

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proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and co-immunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371–445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1−/− neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1−/− neurons. Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.

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5

Lee, Hee Chul, Thomas Hon, Changgui Lan, and Li Zhang. "Structural Environment Dictates the Biological Significance of Heme-Responsive Motifs and the Role of Hsp90 in the Activation of the Heme Activator Protein Hap1." Molecular and Cellular Biology 23, no. 16 (August 15, 2003): 5857–66. http://dx.doi.org/10.1128/mcb.23.16.5857-5866.2003.

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ABSTRACT Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins. The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1. To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1. We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90. Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90. These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes. The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence. Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction. It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation. Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction.

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Xiang, Jianxing, Su Yang, Ning Xin, Marta A. Gaertig, Roger H. Reeves, Shihua Li, and Xiao-Jiang Li. "DYRK1A regulates Hap1–Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome." Proceedings of the National Academy of Sciences 114, no. 7 (January 30, 2017): E1224—E1233. http://dx.doi.org/10.1073/pnas.1614893114.

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Huntingtin-associated protein 1 (Hap1) is known to be critical for postnatal hypothalamic function and growth. Hap1 forms stigmoid bodies (SBs), unique neuronal cytoplasmic inclusions of unknown function that are enriched in hypothalamic neurons. Here we developed a simple strategy to isolate the SB-enriched fraction from mouse brain. By analyzing Hap1 immunoprecipitants from this fraction, we identified a Hap1-interacting SB component, DDB1 and CUL4 associated factor 7 (Dcaf7)/WD40 repeat 68 (WDR68), whose protein level and nuclear translocation are regulated by Hap1. Moreover, we found that Hap1 bound Dcaf7 competitively in cytoplasm with dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a protein implicated in Down syndrome (DS). Depleting Hap1 promoted the DYRK1A–Dcaf7 interaction and increased the DYRK1A protein level. Transgenic DS mice overexpressing DYRK1A showed reduced Hap1–Dcaf7 association in the hypothalamus. Furthermore, the overexpression of DYRK1A in the hypothalamus led to delayed growth in postnatal mice, suggesting that DYRK1A regulates the Hap1–Dcaf7 interaction and postnatal growth and that targeting Hap1 or Dcaf7 could ameliorate growth retardation in DS.

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7

Gong, Yan-Ju, Ying Feng, Yuan-Yuan Cao, Jia Zhao, Wei Wu, Ya-Yun Zheng, Jia-Rui Wu, Xin Li, Gui-Zhi Yang, and Xue Zhou. "Huntingtin-associated protein 1 plays an essential role in the pathogenesis of type 2 diabetes by regulating the translocation of GLUT4 in mouse adipocytes." BMJ Open Diabetes Research & Care 8, no. 1 (October 2020): e001199. http://dx.doi.org/10.1136/bmjdrc-2020-001199.

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ObjectiveGlucose disposal by insulin-responsive tissues maintains the body glucose homeostasis and insulin resistance leads to a risk of developing type 2 diabetes (T2DM). Insulin stimulates the translocation of glucose transporter isoform 4 (GLUT4) vesicles from intracellular compartments to the plasma membrane to facilitate glucose uptake. However, the underlying mechanisms of GLUT4 vesicle translocation are not well defined. Here we show the role of huntingtin-associated protein 1 (HAP1) in GLUT4 translocation in adipocytes and the pathogenesis of T2DM.Research design and methodsThe parameters for glucose metabolism including body weight, glucose tolerance and insulin tolerance were assessed in wild-type (WT) and Hap1+/- mice. HAP1 protein expression was verified in adipose tissue. Hap1 mRNA and protein expression was monitored in adipose tissue of high-fat diet (HFD)-induced diabetic mice. Insulin-stimulated GLUT4 vesicle translocation and glucose uptake were detected using immunofluorescence techniques and quantified in primary adipocytes from Hap1-/- mice. The interaction between HAP1 and GLUT4 was assessed by immunofluorescence colocalization and co-immunoprecipitation in HEK293 cells and adipose tissue. The role of sortilin in HAP1 and GLUT4 interaction was approved by co-immunoprecipitation and RNA interference.ResultsThe expression of Hap1 mRNA and protein was detected in WT mouse adipose tissue and downregulated in adipose tissue of HFD-induced diabetic mice. Hap1+/- mice exhibited increased body weight, pronounced glucose tolerance and significant insulin intolerance compared with the WT mice. HAP1 colocalized with GLUT4 in mouse adipocytes and cotransfected HEK293 cells. Furthermore, the insulin-stimulated GLUT4 vesicle translocation and glucose uptake were defective in Hap1-/- adipocytes. Finally, sortilin mediated the interaction of HAP1 and GLUT4.ConclusionsOur study showed that HAP1 formed a protein complex with GLUT4 and sortilin, and played a critical role in insulin-stimulated GLUT4 translocation in adipocytes. Its downregulation may contribute to the pathogenesis of diabetes.

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8

Hach, Angela, Thomas Hon, and Li Zhang. "A New Class of Repression Modules Is Critical for Heme Regulation of the Yeast Transcriptional Activator Hap1." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4324–33. http://dx.doi.org/10.1128/mcb.19.6.4324.

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ABSTRACTHeme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeastSaccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules—RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244—is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

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9

Hickman, Mark J., and Fred Winston. "Heme Levels Switch the Function of Hap1 of Saccharomyces cerevisiae between Transcriptional Activator and Transcriptional Repressor." Molecular and Cellular Biology 27, no. 21 (September 4, 2007): 7414–24. http://dx.doi.org/10.1128/mcb.00887-07.

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ABSTRACT Changes in oxygen levels cause widespread changes in gene expression in organisms ranging from bacteria to humans. In Saccharomyces cerevisiae, this response is mediated in part by Hap1, originally identified as a heme-dependent transcriptional activator that functions during aerobic growth. We show here that Hap1 also plays a significant and direct role under hypoxic conditions, not as an activator, but as a repressor. The repressive activity of Hap1 controls several genes, including three ERG genes required for ergosterol biosynthesis. Chromatin immunoprecipitation experiments showed that Hap1 binds to the ERG gene promoters, while additional experiments showed that the corepressor Tup1/Ssn6 is recruited by Hap1 and is also required for repression. Furthermore, mutational analysis demonstrated that conserved Hap1 binding sites in the ERG5 5′ regulatory region are required for repression. The switch of Hap1 from acting as a hypoxic repressor to an aerobic activator is determined by heme, which is synthesized only in the presence of oxygen. The ability of Hap1 to function as a ligand-dependent repressor and activator is a property shared with mammalian nuclear hormone receptors and likely allows greater transcriptional control by Hap1 in response to changing oxygen levels.

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Hon, Thomas, Hee Chul Lee, Angela Hach, Jill L. Johnson, Elizabeth A. Craig, Hediye Erdjument-Bromage, Paul Tempst, and Li Zhang. "The Hsp70-Ydj1 Molecular Chaperone Represses the Activity of the Heme Activator Protein Hap1 in the Absence of Heme." Molecular and Cellular Biology 21, no. 23 (December 1, 2001): 7923–32. http://dx.doi.org/10.1128/mcb.21.23.7923-7932.2001.

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ABSTRACT In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

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11

Zhang, Li, Angela Hach, and Cheng Wang. "Molecular Mechanism Governing Heme Signaling in Yeast: a Higher-Order Complex Mediates Heme Regulation of the Transcriptional Activator HAP1." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3819–28. http://dx.doi.org/10.1128/mcb.18.7.3819.

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ABSTRACT Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization. To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast heme-responsive transcriptional activator HAP1. HAP1 activity is regulated precisely and tightly by heme. Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex. Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1. The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity. Further, we determined HAP1 domains required for heme regulation: three domains—the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain—cooperate to form the higher-order complex and mediate heme regulation. Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation. Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

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Ushinsky, S. C., and T. Keng. "A novel allele of HAP1 causes uninducible expression of HEM13 in Saccharomyces cerevisiae." Genetics 136, no. 3 (March 1, 1994): 819–31. http://dx.doi.org/10.1093/genetics/136.3.819.

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Abstract Transcription of HEM13 in Saccharomyces cerevisiae is repressed by heme and oxygen. We have isolated two mutants in which expression of HEM13 is aberrant. The mutant alleles in these strains represent two different alleles of HAP1. HAP1 encodes an activator protein whose DNA binding activity is stimulated by heme, and is required for the transcription of CYC1, ROX1 and a number of other heme-dependent genes. One of our mutant alleles confers a phenotype much like that of the hap1::LEU2 allele. Expression of HEM13 in a strain with this mutation is elevated under repressing conditions and not fully inducible in the absence of heme. The other mutant allele of HAP1 we uncovered confers a novel phenotype. A strain containing this allele exhibits heme-independent expression of CYC1 and ROX1 and uninducible expression of HEM13 and ANB1. The mutation associated with this novel allele of HAP1 was localized to a glycine to aspartate change in amino acid 235 of HAP1, between the DNA binding and heme responsive domains. DNA binding assays demonstrated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme.

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Liao, Min, Jianying Shen, Yinong Zhang, Shi-Hua Li, Xiao-Jiang Li, and He Li. "Immunohistochemical Localization of Huntingtin-associated Protein 1 in Endocrine System of the Rat." Journal of Histochemistry & Cytochemistry 53, no. 12 (June 27, 2005): 1517–24. http://dx.doi.org/10.1369/jhc.5a6662.2005.

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Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.

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Zhang, L., and L. Guarente. "Evidence that TUP1/SSN6 has a positive effect on the activity of the yeast activator HAP1." Genetics 136, no. 3 (March 1, 1994): 813–17. http://dx.doi.org/10.1093/genetics/136.3.813.

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Abstract The activity of the yeast transcriptional activator HAP1 is controlled by heme and the heme effect is mediated through the heme domain of HAP1. In this report, we show that HAP1 activity is significantly reduced in strains deleted of TUP1 or SSN6, and addition of a heme analog does not allow HAP1 to regain its full activity. Deletion of the heme domain alleviates the requirement for TUP1/SSN6. The results suggest that TUP1/SSN6 have a positive effect on the activity of HAP1 and this effect is mediated through the heme domain. Although TUP1/SSN6 generally repress transcription of many genes, our data indicate they may have positive effect on the expression of certain genes.

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Keng, T. "HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 6 (June 1992): 2616–23. http://dx.doi.org/10.1128/mcb.12.6.2616.

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HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.

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Keng, T. "HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae." Molecular and Cellular Biology 12, no. 6 (June 1992): 2616–23. http://dx.doi.org/10.1128/mcb.12.6.2616-2623.1992.

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HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.

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Liu, Li, Mengdi Liu, Di Zhang, Shanjun Deng, Piaopiao Chen, Jing Yang, Yunhan Xie, and Xionglei He. "Decoupling gene functions from knockout effects by evolutionary analyses." National Science Review 7, no. 7 (April 24, 2020): 1169–80. http://dx.doi.org/10.1093/nsr/nwaa079.

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Abstract Genic functions have long been confounded by pleiotropic mutational effects. To understand such genetic effects, we examine HAP4, a well-studied transcription factor in Saccharomyces cerevisiae that functions by forming a tetramer with HAP2, HAP3 and HAP5. Deletion of HAP4 results in highly pleiotropic gene expression responses, some of which are clustered in related cellular processes (clustered effects) while most are distributed randomly across diverse cellular processes (distributed effects). Strikingly, the distributed effects that account for much of HAP4 pleiotropy tend to be non-heritable in a population, suggesting they have few evolutionary consequences. Indeed, these effects are poorly conserved in closely related yeasts. We further show substantial overlaps of clustered effects, but not distributed effects, among the four genes encoding the HAP2/3/4/5 tetramer. This pattern holds for other biochemically characterized yeast protein complexes or metabolic pathways. Examination of a set of cell morphological traits of the deletion lines yields consistent results. Hence, only some deletion effects of a gene support related biochemical understandings with the rest being often pleiotropic and evolutionarily decoupled from the gene's normal functions. This study suggests a new framework for reverse genetic analysis.

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Lee, Jung Kwon, SungMyung Kang, Xidi Wang, Jesusa L. Rosales, Xu Gao, Hee-Guk Byun, Yan Jin, Songbin Fu, Jinghua Wang, and Ki-Young Lee. "HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway." Blood 133, no. 20 (May 16, 2019): 2222–32. http://dx.doi.org/10.1182/blood-2018-12-890236.

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Abstract l-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3. Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase–induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

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Beigl, Tobias B., Ine Kjosås, Emilie Seljeseth, Nina Glomnes, and Henriette Aksnes. "Efficient and crucial quality control of HAP1 cell ploidy status." Biology Open 9, no. 11 (November 12, 2020): bio057174. http://dx.doi.org/10.1242/bio.057174.

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ABSTRACTThe near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.

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Chantrel, Yann, Mauricette Gaisne, Claire Lions, and Jacqueline Verdière. "The Transcriptional Regulator Hap1p (Cyp1p) Is Essential for Anaerobic or Heme-Deficient Growth of Saccharomyces cerevisiae: Genetic and Molecular Characterization of an Extragenic Suppressor that Encodes a WD Repeat Protein." Genetics 148, no. 2 (February 1, 1998): 559–69. http://dx.doi.org/10.1093/genetics/148.2.559.

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Abstract We report here that Hap1p (originally named Cyp1p) has an essential function in anaerobic or heme-deficient growth. Analysis of intragenic revertants shows that this function depends on the amino acid preceding the first cysteine residue of the DNA-binding domain of Hap1p. Selection of recessive extragenic suppressors of a hap1−hem1− strain allowed the identification, cloning, and molecular analysis of ASC1 (Cyp1 Absence of growth Supressor). The sequence of ASC1 reveals that its ORF is interrupted by an intron that shelters the U24 snoRNA. Deletion of the intron, inactivation of the ORF, and molecular localization of the mutations show unambiguously that it is the protein and not the snoRNA that is involved in the suppressor phenotype. ASC1, which is constitutively transcribed, encodes an abundant, cytoplasmically localized 35-kD protein that belongs to the WD repeat family, which is found in a large variety of eucaryotic organisms. Polysome profile analysis supports the involvement of this protein in translation. We propose that the absence of functional Asc1p allows the growth of hap1−hem1− cells by reducing the efficiency of translation. Based on sequence comparisons, we discuss the possibility that the protein intervenes in a kinase-dependent signal transduction pathway involved in this last function.

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Liu, Qiong, Siying Cheng, Huiming Yang, Louyin Zhu, Yongcheng Pan, Liang Jing, Beisha Tang, Shihua Li, and Xiao-Jiang Li. "Loss of Hap1 selectively promotes striatal degeneration in Huntington disease mice." Proceedings of the National Academy of Sciences 117, no. 33 (August 3, 2020): 20265–73. http://dx.doi.org/10.1073/pnas.2002283117.

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Huntington disease (HD) is an ideal model for investigating selective neurodegeneration, as expanded polyQ repeats in the ubiquitously expressed huntingtin (HTT) cause the preferential neurodegeneration in the striatum of the HD patient brains. Here we report that adeno-associated virus (AAV) transduction-mediated depletion of Hap1, the first identified huntingtin-associated protein, in adult HD knock-in (KI) mouse brains leads to selective neuronal loss in the striatum. Further, Hap1 depletion-mediated neuronal loss via AAV transduction requires the presence of mutant HTT. Rhes, a GTPase that is enriched in the striatum and sumoylates mutant HTT to mediate neurotoxicity, binds more N-terminal HTT when Hap1 is deficient. Consistently, more soluble and sumoylated N-terminal HTT is presented in HD KI mouse striatum when HAP1 is absent. Our findings suggest that both Rhes and Hap1 as well as cellular stress contribute to the preferential neurodegeneration in HD, highlighting the involvement of multiple factors in selective neurodegeneration.

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Li, Xiao-Jiang, and Shi-Hua Li. "HAP1 and intracellular trafficking." Trends in Pharmacological Sciences 26, no. 1 (January 2005): 1–3. http://dx.doi.org/10.1016/j.tips.2004.11.001.

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23

Li, Shi-Hua, Seyed H. Hosseini, Claire-Anne Gutekunst, Steven M. Hersch, Robert J. Ferrante, and Xiao-Jiang Li. "A Human HAP1 Homologue." Journal of Biological Chemistry 273, no. 30 (July 24, 1998): 19220–27. http://dx.doi.org/10.1074/jbc.273.30.19220.

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24

Lombardía, L. J., J. L. Cadahía-Rodríguez, M. A. Freire-Picos, M. I. González-Siso, A. M. Rodríguez-Torres, and M. E. Cerdán. "Transcript analysis of 203 novel genes from Saccharomyces cerevisiae in hap1 and rox1 mutant backgrounds." Genome 43, no. 5 (October 1, 2000): 881–86. http://dx.doi.org/10.1139/g00-049.

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Hap1 and Rox1 are transcriptional regulators that bind regulatory sites in the promoters of oxygen-regulated genes in Saccharomyces cerevisiae. Hap1 is a heme-responsive activator of genes induced in aerobic conditions and Rox1 is a repressor of hypoxic genes in aerobic conditions. We have studied transcriptional regulation of a pool of 203 open reading frames (ORFs) from chromosomes IV, VII, and XIV in wild-type, hap1, and rox1 mutant genetic backgrounds in an attempt to extend the family of oxygen and heme regulated genes. Only three ORFs are significantly repressed by Rox1 but they cannot be considered as typical hypoxic genes because they are not overexpressed during hypoxia.Key words: Saccharomyces cerevisiae, genome analysis, chromosomes IV, VII, and XIV, gene expression, ROX1 and HAP1.

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Walker, L. J., C. N. Robson, E. Black, D. Gillespie, and I. D. Hickson. "Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding." Molecular and Cellular Biology 13, no. 9 (September 1993): 5370–76. http://dx.doi.org/10.1128/mcb.13.9.5370.

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The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

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Walker, L. J., C. N. Robson, E. Black, D. Gillespie, and I. D. Hickson. "Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding." Molecular and Cellular Biology 13, no. 9 (September 1993): 5370–76. http://dx.doi.org/10.1128/mcb.13.9.5370-5376.1993.

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The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

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27

Bao, Wei-Guo, Bernard Guiard, Zi-An Fang, Claudia Donnini, Michel Gervais, Flavia M. Lopes Passos, Iliana Ferrero, Hiroshi Fukuhara, and Monique Bolotin-Fukuhara. "Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis." Eukaryotic Cell 7, no. 11 (September 19, 2008): 1895–905. http://dx.doi.org/10.1128/ec.00018-08.

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ABSTRACT The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

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Prezant, T., K. Pfeifer, and L. Guarente. "Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation." Molecular and Cellular Biology 7, no. 9 (September 1987): 3252–59. http://dx.doi.org/10.1128/mcb.7.9.3252.

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Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

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Prezant, T., K. Pfeifer, and L. Guarente. "Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation." Molecular and Cellular Biology 7, no. 9 (September 1987): 3252–59. http://dx.doi.org/10.1128/mcb.7.9.3252-3259.1987.

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Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

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30

Bourne, Christina R., M. G. Finn, and Adam Zlotnick. "Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1." Journal of Virology 80, no. 22 (August 30, 2006): 11055–61. http://dx.doi.org/10.1128/jvi.00933-06.

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ABSTRACT Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy.

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Rong, Juan, Shi-Hua Li, and Xiao-Jiang Li. "Regulation of intracellular HAP1 trafficking." Journal of Neuroscience Research 85, no. 14 (2007): 3025–29. http://dx.doi.org/10.1002/jnr.21326.

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32

Olesen, J. T., J. D. Fikes, and L. Guarente. "The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain." Molecular and Cellular Biology 11, no. 2 (February 1991): 611–19. http://dx.doi.org/10.1128/mcb.11.2.611.

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The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

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Olesen, J. T., J. D. Fikes, and L. Guarente. "The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain." Molecular and Cellular Biology 11, no. 2 (February 1991): 611–19. http://dx.doi.org/10.1128/mcb.11.2.611-619.1991.

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The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

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34

Rapuntean, Sorin, Alexandru Pop, Vasile Miclaus, Corina Garbo, Flore Chirila, Gheorghe Rapuntean, Nicodim Fit, Horea Farcau, and Maria Tomoaia-Cotisel. "In Vitro Sensitivity Research Concerning Some Microorganisms at Hydroxyquinoline and Cupric Derivatives Deposited onto Hydroxyapatite." Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Veterinary Medicine 73, no. 2 (November 30, 2016): 339. http://dx.doi.org/10.15835/buasvmcn-vm:12203.

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ABSTRACT Introduction: The preparations based on hydroxyquinoline, in various combinations, are used in medicine, being shown to have an inhibitory effect against bacteria, molds, fungi, parasites, and viruses, but also having other beneficial effects mentioned in other medical conditions (anti-cancer, anti-degenerative, anti-inflammatory). Aims: In vitro susceptibility testing of microorganisms: bacteria (Gram positive and Gram negative), yeast (Candida spp.,) and unicellular algae (Prototheca spp.) at the preparations based on hydroxyquinoline (HQ) and its cupric derivatives deposited on hydroxyapatite (HAP). Materials and methods: There were tested microbial strains of the following genera: Escherichia, Staphylococcus, Micrococcus, Bacillus, Candida, and Prototheca. The tested products (developed in the Laboratory for Nanobiomaterials Synthesis, Center of Physical Chemistry, Faculty of Chemistry and Chemical Engineering, UBB Cluj-Napoca) were developed in three versions: 1) HQ–Cu2+–HAP1; 2) HQ–Cu2+–HAP2; and 3) NHQ–Cu2+–HAP2, where NHQ stands for nitro hydroxyquinoline. Determination of the inhibitory effect was conducted by diffusion technique in nutrient agar, according to CLSI 2013 standards, with necessary adaptations for testing of products made in the form of suspensions. Results: Following interpretation, it was found that the inhibition zones, arising from the antimicrobial effect of the tested products showed variability in size, depending on the test product and the microbial strain: Escherichia coli (8-10 mm), Staphylococcus sp. (17.6 - 23.2 mm), Micrococcus spp. (24.4 - 27.6 mm), Bacillus spp. (14.0 - 16.0 mm), Candida spp. (20.4 - 25.2 mm), Prototheca spp. (20.8 - 30.0 mm). From the three tested products, the best efficacy was found at the product no. 3 (NHQ – Cu2+ – HAP2), followed by no. 1 (HQ– Cu2+–HAP1) and no. 2 (HQ–Cu2+–HAP2). Conclusions: The inhibitory effect was bactericidal, manifested more intensively against Gram-positive bacteria, yeasts, and prototheca. Such products, prepared in the form of suspensions, may have practical application in the prevention and treatment of skin or hooves disorders. No resistance phenomena are recorded. Keywords: copper, hydroxyapatite, hydroxyquinoline, microorganisms, sensitivity.

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35

Gutekunst, Claire-Anne, Enrique R. Torre, Zejuan Sheng, Hong Yi, Sarah H. Coleman, I. Björn Riedel, and Hideaki Bujo. "Stigmoid Bodies Contain Type I Receptor Proteins SorLA/LR11 and Sortilin: New Perspectives on Their Function." Journal of Histochemistry & Cytochemistry 51, no. 6 (June 2003): 841–52. http://dx.doi.org/10.1177/002215540305100615.

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Stigmoid bodies (SBs) are structures in the cytoplasm of neurons. SBs are mostly found in the hypothalamic region of the rat and contain a protein called huntingtin-associated protein 1 (HAP1). In a recent publication, large cytoplasmic structures were shown to be immunoreactive for a type I receptor called SorLA/LR11. By light microscopic analysis, these structures appeared similar to SBs in size and in brain regional and subcellular localization. To determine whether these large puncta correspond to HAP1-containing SBs, we used antibodies specific to various domains of the apolipoprotein receptor LR11 to perform immunocytochemistry in rat and mouse brain tissue. Transfection studies using HeLa cells were conducted to demonstrate the specificity of the antibodies. We found that, in both species, antibodies to the domain II (or VSP10 for vacuolar sorting protein 10 domain) of LR11 immunoreact with large cytoplasmic structures. Co-localization immunolabeling experiments in rat brain tissue sections and in neuron cultures showed that these LR11-immunoreactive structures correspond to HAP1-positive SBs. Electron microscopy was performed in rat hypothalamus and further demonstrated the presence of LR11 in SBs and its co-localization with HAP1. LR11-containing SBs were most abundant in the hypothalamus but were also found in many brainstem nuclei, thalamus, and hippocampus. Our data also show that sortilin, another transmembrane protein containing a VPS10 domain, localizes to large cytoplasmic puncta and is found in LR11-positive and Hap1-positive SBs in hypothalamic neuron cultures.

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Muramoto, Kazumasa, Shigeru Makishima, Shin-Ichi Aizawa, and Robert M. Macnab. "Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook ofSalmonella." Journal of Bacteriology 181, no. 18 (September 15, 1999): 5808–13. http://dx.doi.org/10.1128/jb.181.18.5808-5813.1999.

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ABSTRACT The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, afliC (flagellin) mutant, or a fliD(hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

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Lai, Liang-Chuan, Alexander L. Kosorukoff, Patricia V. Burke, and Kurt E. Kwast. "Metabolic-State-Dependent Remodeling of the Transcriptome in Response to Anoxia and Subsequent Reoxygenation in Saccharomyces cerevisiae." Eukaryotic Cell 5, no. 9 (September 2006): 1468–89. http://dx.doi.org/10.1128/ec.00107-06.

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ABSTRACT We conducted a comprehensive genomic analysis of the temporal response of yeast to anaerobiosis (six generations) and subsequent aerobic recovery (≈2 generations) to reveal metabolic-state (galactose versus glucose)-dependent differences in gene network activity and function. Analysis of variance showed that far fewer genes responded (raw P value of ≤10−8) to the O2 shifts in glucose (1,603 genes) than in galactose (2,388 genes). Gene network analysis reveals that this difference is due largely to the failure of “stress”-activated networks controlled by Msn2/4, Fhl1, MCB, SCB, PAC, and RRPE to transiently respond to the shift to anaerobiosis in glucose as they did in galactose. After ≈1 generation of anaerobiosis, the response was similar in both media, beginning with the deactivation of Hap1 and Hap2/3/4/5 networks involved in mitochondrial functions and the concomitant derepression of Rox1-regulated networks for carbohydrate catabolism and redox regulation and ending (≥2 generations) with the activation of Upc2- and Mot3-regulated networks involved in sterol and cell wall homeostasis. The response to reoxygenation was rapid (<5 min) and similar in both media, dominated by Yap1 networks involved in oxidative stress/redox regulation and the concomitant activation of heme-regulated ones. Our analyses revealed extensive networks of genes subject to combinatorial regulation by both heme-dependent (e.g., Hap1, Hap2/3/4/5, Rox1, Mot3, and Upc2) and heme-independent (e.g., Yap1, Skn7, and Puf3) factors under these conditions. We also uncover novel functions for several cis-regulatory sites and trans-acting factors and define functional regulons involved in the physiological acclimatization to changes in oxygen availability.

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Sočo, Eleonora, Dorota Papciak, Magdalena M. Michel, Dariusz Pająk, Andżelika Domoń, and Bogdan Kupiec. "Characterization of the Physical, Chemical, and Adsorption Properties of Coal-Fly-Ash–Hydroxyapatite Composites." Minerals 11, no. 7 (July 16, 2021): 774. http://dx.doi.org/10.3390/min11070774.

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(1) Hydroxyapatite (HAp), which can be obtained by several methods, is known to be a good adsorbent. Coal fly ash (CFA) is a commonly reused byproduct also used in environmental applications as an adsorbent. We sought to answer the following question: Can CFA be included in the method of HAp wet synthesis to produce a composite capable of adsorbing both heavy metals and dyes? (2) High calcium lignite CFA from the thermal power plant in Bełchatów (Poland) was used as the base to prepare CFA–HAp composites. Four types designated CFA–HAp1–4 were synthesized via the wet method of in situ precipitation. The synthesis conditions differed in terms of the calcium reactants used, pH, and temperature. We also investigated the equilibrium adsorption of Cu(II) and rhodamine B (RB) on CFA–HAp1–4. The data were fitted using the Langmuir, Freundlich, and Redlich–Peterson models and validated using R2 and χ2/DoF. Surface changes in CFA–HAp2 following Cu(II) and RB adsorption were assessed using SEM, SE, and FT-IR analysis. (3) The obtained composites contained hydroxyapatite (Ca/P 1.67) and aluminosilicates. The mode of Cu(II) and RB adsorption could be explained by the Redlich–Peterson model. The CFA–HAp2 obtained using CFA, Ca(NO3)2, and (NH4)2HPO4 at RT and pH 11 exhibited the highest maximal adsorption capacity: 73.6 mg Cu/g and 87.0 mg RB/g. (4) The clear advantage of chemisorption over physisorption was indicated by the Cu(II)–CFA–HAp system. The RB molecules present in the form of uncharged lactone were favorably adsorbed even on strongly deprotonated CFA–HAp surfaces.

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Maggi, Maristella, and Claudia Scotti. "HAP1 loss in l-asparaginase resistance." Blood 133, no. 20 (May 16, 2019): 2116–18. http://dx.doi.org/10.1182/blood-2019-03-900993.

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40

Kirchhoff, Aaron M., Eric L. Barker, and Julia A. Chester. "Endocannabinoids and Fear-Related Behavior in Mice Selectively Bred for High or Low Alcohol Preference." Brain Sciences 9, no. 10 (September 26, 2019): 254. http://dx.doi.org/10.3390/brainsci9100254.

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Alcohol use disorders (AUDs) have a high incidence of co-morbidity with stress-related psychopathologies, such as post-traumatic stress disorder (PTSD). Genetic and pharmacological studies support a prominent role for the endocannabinoid system (ECS) in modulating stress-related behaviors relevant to AUDs and PTSD. Mouse lines selectively bred for high (HAP) and low (LAP) alcohol preference show reproducible differences in fear-potentiated startle (FPS), a model for PTSD-related behavior. The first experiment in this study assessed levels of the endocannabinoids, anandamide (AEA) and sn-2 arachidonylglycerol (2-AG), in the prefrontal cortex (PFC), amygdala (AMG), and hippocampus (HIP) of male and female HAP1 and LAP1 mice following the expression of FPS to determine whether ECS responses to conditioned-fear stress (FPS) were correlated with genetic propensity toward high or low alcohol preference. The second experiment examined effects of a cannabinoid receptor type 1 agonist (CP55940) and antagonist (rimonabant) on the expression of FPS in HAP1 and LAP1 male and female mice. The estrous cycle of females was monitored throughout the experiments to determine if the expression of FPS differed by stage of the cycle. FPS was greater in male and female HAP1 than LAP1 mice, as previously reported. In both experiments, LAP1 females in diestrus displayed greater FPS than LAP1 females in metestrus and estrus. In the AMG and HIP, AEA levels were greater in male fear-conditioned HAP1 mice than LAP1 mice. There were no line or sex differences in effects of CP55940 or rimonabant on the expression of FPS. However, surprisingly, evidence for anxiogenic effects of prior treatment with CP55940 were seen in all mice during the third drug-free FPS test. These findings suggest that genetic differences in ECS function in response to fear-conditioning stress may underlie differences in FPS expression in HAP1 and LAP1 selected lines.

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Branger, C., C. Sonrier, B. Chatrenet, B. Klonjkowski, N. Ruvoen-Clouet, A. Aubert, G. André-Fontaine, and M. Eloit. "Identification of the Hemolysis-Associated Protein 1 as a Cross-Protective Immunogen of Leptospira interrogans by Adenovirus-Mediated Vaccination." Infection and Immunity 69, no. 11 (November 1, 2001): 6831–38. http://dx.doi.org/10.1128/iai.69.11.6831-6838.2001.

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ABSTRACT New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membraneLeptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

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Branger, C., B. Chatrenet, A. Gauvrit, F. Aviat, A. Aubert, J. M. Bach, and G. André-Fontaine. "Protection against Leptospira interrogans Sensu Lato Challenge by DNA Immunization with the Gene Encoding Hemolysin-Associated Protein 1." Infection and Immunity 73, no. 7 (July 2005): 4062–69. http://dx.doi.org/10.1128/iai.73.7.4062-4069.2005.

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ABSTRACT The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.

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43

Liu, Xiaoli, Yun Li, Laiyuan Wang, Qi Zhao, Xiangfeng Lu, Jianfeng Huang, Zhongjie Fan, and Dongfeng Gu. "The INSIG1 gene, not the INSIG2 gene, associated with coronary heart disease: tagSNPs and haplotype-based association study." Thrombosis and Haemostasis 100, no. 05 (2008): 886–92. http://dx.doi.org/10.1160/th08-01-0050.

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SummaryDyslipidemia, especially hypercholesterolemia, is a major risk factor of coronary heart disease (CHD). The insulin induced gene 1 (INSIG1) and insulin induced gene 2 (INSIG2) encode two proteins which mediate feedback control of cholesterol synthesis. We conducted a case-control study to investigate whether the common variants in INSIG genes were associated with CHD in Chinese Han population. Three single nucleotide polymorphisms (SNPs) of the INSIG1 gene and four SNPs of the INSIG2 gene were chosen as haplotype-tagging SNPs (htSNPs) and genotyped in 853 patients with CHD and 948 unrelated control subjects. Haplotype analysis showed that the haplotype Hap4 (TTA) of the INSIG1 gene significantly increased the risk of CHD (adjusted odds ratio [OR]1.59, 95% confidence interval [CI] 1.22–2.06,p=0.0006),while the haplotype Hap3 (TGA) significantly decreased the risk of CHD (adjusted OR 0.74, 95%CI 0.60–0.92, p=0.006) compared with the reference haplotype Hap1 (GGA). No significant associations were found between polymorphisms of INSIG2 gene and CHD. In addition, the single polymorphism analysis showed that rs9769826 of the INSIG1 gene was associated with glucose in controls. The G-allele (minor allele) carriers had higher glucose level (5.74 ± 2.03 mM) than AA genotype carriers (5.45 ± 1.37 mM,p=0.015).The present study indicated that the INSIG1 gene, but not the INSIG2 gene, was associated with CHD in the Chinese population.

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44

Li, Shihua, and Xiao-Jiang Li. "A huntingtin–HAP1–PCM1 pathway in ciliogenesis." Expert Review of Proteomics 9, no. 1 (February 2012): 17–19. http://dx.doi.org/10.1586/epr.11.72.

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45

Woods, Stephen C., and Randy J. Seeley. "Hap1 and GABA: Thinking about food intake." Cell Metabolism 3, no. 6 (June 2006): 388–90. http://dx.doi.org/10.1016/j.cmet.2006.05.007.

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Thorsness, M., W. Schafer, L. D'Ari, and J. Rine. "Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae." Molecular and Cellular Biology 9, no. 12 (December 1989): 5702–12. http://dx.doi.org/10.1128/mcb.9.12.5702.

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Responses of the yeast genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMG1 and HMG2, to in vivo changes in heme concentrations were investigated. Expression of the genes was determined by direct measurement of the mRNA transcribed from each gene, by direct assay of the enzyme activity encoded by each gene, and by measurement of the expression of lacZ fusions to the control regions of each gene. These studies indicated that expression of HMG1 was stimulated by heme, whereas expression of HMG2 was repressed by heme. The effect of heme on HMG1 expression was mediated by the HAP1 transcriptional regulator and was independent of HAP2. Thus, the genes encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase isozymes join a growing list of gene pairs that are regulated by heme in opposite ways.

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47

Thorsness, M., W. Schafer, L. D'Ari, and J. Rine. "Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae." Molecular and Cellular Biology 9, no. 12 (December 1989): 5702–12. http://dx.doi.org/10.1128/mcb.9.12.5702-5712.1989.

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Responses of the yeast genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMG1 and HMG2, to in vivo changes in heme concentrations were investigated. Expression of the genes was determined by direct measurement of the mRNA transcribed from each gene, by direct assay of the enzyme activity encoded by each gene, and by measurement of the expression of lacZ fusions to the control regions of each gene. These studies indicated that expression of HMG1 was stimulated by heme, whereas expression of HMG2 was repressed by heme. The effect of heme on HMG1 expression was mediated by the HAP1 transcriptional regulator and was independent of HAP2. Thus, the genes encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase isozymes join a growing list of gene pairs that are regulated by heme in opposite ways.

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Meira, Lisiane B., David L. Cheo, Robert E. Hammer, Dennis K. Burns, Antonio Reis, and Errol C. Friedberg. "Genetic interaction between HAP1/REF-1 and p53." Nature Genetics 17, no. 2 (October 1997): 145. http://dx.doi.org/10.1038/ng1097-145.

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Pfeifer, Karl, Kwang-Soo Kim, Scott Kogan, and Leonard Guarente. "Functional dissection and sequence of yeast HAP1 activator." Cell 56, no. 2 (January 1989): 291–301. http://dx.doi.org/10.1016/0092-8674(89)90903-3.

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Lodi, T., and B. Guiard. "Complex transcriptional regulation of the Saccharomyces cerevisiae CYB2 gene encoding cytochrome b2: CYP1(HAP1) activator binds to the CYB2 upstream activation site UAS1-B2." Molecular and Cellular Biology 11, no. 7 (July 1991): 3762–72. http://dx.doi.org/10.1128/mcb.11.7.3762.

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Expression of the Saccharomyces cerevisiae gene encoding cytochrome b2 (EC 1.2.2.3), CYB2, was investigated by direct analysis of mRNA transcripts and by measurement of the expression of lacZ fused to the CYB2 control regions. These studies indicated that regulation of the CYB2 gene is subject to several metabolic controls at the transcriptional level: inhibition due to glucose fermentation, induction by lactate, and inhibition in anaerobiosis or in absence of heme biosynthesis. Furthermore, we have shown that the CYB2 promoter contains one cis negative regulatory region and two heme-dependent positive regions, one of which is controlled by the transcriptional regulator CYP1 (HAP1) which is involved in the modulation of the expression of several oxygen-regulated genes. The CYP1 (HAP1)-binding sequence was located by gel retardation and DNase I footprinting experiments and compared with the binding sequences previously characterized in detail (UAS1CYC1, UAS'CYP3 (CYC7), and UASCTT1).

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Journal articles on the topic 'HAP1'

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