bioRxiv (Cold Spring Harbor Laboratory), Mar 20, 2024
The initiation of microtubule formation in eukaryotes is primarily facilitated by γ-tubulin and t... more The initiation of microtubule formation in eukaryotes is primarily facilitated by γ-tubulin and the highly conserved protein complex called γ-Tubulin Ring Complex (γ-TuRC) in various microtubule-organizing centers (MTOCs). Non-centrosomal MTOCs have a range of functions and contribute to diverse cellular activities in different cell types. While the heterogeneity of tissue-specific MTOCs and γ-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. In this study, we investigate the testis-specific distribution of the canonical γ-TuRC by in vivo tagging of two complex members, Grip163 and Grip84. We find that both the testis-specific (t-γ-TuRC) and canonical form of γ-TuRC are simultaneously present and colocalize with each other at the basal body, apical nuclear tip, and near the elongating mitochondrial derivatives in the post-meiotic spermatids. We propose the possibility of the formation of MTOCs containing both t-γ-TuRC and γ-TuRC complexes. We also find some of the known γ-TuRC binding partners localizing to the apical nuclear tip. Additionally, we identify the Golgi localization of Grip84 in spermatocytes, alongside its centrosomal and diverse post-meiotic localization. These results highlight and prove the importance of the different γ-TuRCs in organizing the diverse MTOCs during the extensive rearrangement of cell organelles in Drosophila spermatogenesis. .
International journal of molecular sciences, Apr 3, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Muc... more Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Advances in Clinical and Experimental Medicine, Aug 24, 2022
There are numerous surprising discoveries in current comprehensive biopolymer research, including... more There are numerous surprising discoveries in current comprehensive biopolymer research, including the description of new types of biopolymers and the extension of their applications. The discovery of a new rotiferspecific biopolymer family (Rotimers) and the exceptional ability of these micrometazoans to inactivate and catabolize human-type neurotoxic aggregates (e.g., beta-amyloids, alpha-synucleins, prions) by their exudates can be mentioned as the original work of our research group. Rotimers are exogenous and protein complex molecules with a calcium-dependent production mechanism in both bdelloid and monogonant rotifers. However, their experimental and application possibilities are still unknown; only part of the class has been explored and described. Current Rotimer-related studies present promising biodiversity and bioactivity of these biomaterials (e.g., anti-and disaggregation effects or high degrees of adhesion to other molecules). The primary objective of current research is to explore and develop their application in translational biomedicine. A key area is the design of drug candidates against neurodegeneration-related aggregates based on the molecular information provided by the composition, structure and function of Rotimers. These novel biomaterials have the potential to open new perspectives in the pharmaceutical industry and healthcare.
The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effec... more The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, we used a comprehensive set of Drosophila Rabs for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. We confirm over 25 interactions for Rab2,
Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. The... more Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrial derivatives in spermatids. Testis-specific genes are less conserved and could gain new functions, thus raising a question whether Bb8 has retained its original enzymatic activity. We show that while Bb8 displays glutamate dehydrogenase activity, there are significant functional differences between the housekeeping Gdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8ms mutant phenotype, with superior performance by GLUD2. We also tested the role of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of our study indicate that Droso...
Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking acti... more Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin.
Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria fr... more Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates-both from keratomycosis and environment-were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.
bioRxiv (Cold Spring Harbor Laboratory), May 31, 2023
Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, whi... more Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which result in similarly abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms and interrelationships of PP4 and Ctb in the regulation of HR repair.
Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogen... more Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of SLap proteins we show the characterization of classical mutants and RNAi lines affecting of the SLap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the SLap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that SLap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that SLap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.
International Journal of Molecular Sciences, Jan 19, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelag... more The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology. Drosophila melanogaster testis is a particularly suitable model to follow the process of spermatogenesis 1,2. During Drosophila spermatogenesis from a single spermatogonia sixty-four sperms are being produced, through mitotic and meiotic cell divisions and intensive cellular remodeling. After each division, the daughter cells remain in connection with plasma bridges, which allows them to develop simultaneously in a syncytial cyst. The early transitamplifying mitotic divisions result in sixteen spermatocytes. Spermatocytes undergo a maturing process, they have high transcriptional activity until meiosis and the accumulated transcripts contribute to the development of the transcriptionally mostly inactive post-meiotic stages 3. After meiosis the mitochondria aggregate and fuse to establish the nebenkern structure, which consists of two mitochondrial derivatives. Next, the spermatids start to elongate to reach a full length of ~ 1.8 mm, which approximately 150 times longer than their initial diameter. The two mitochondrial derivatives run along the axoneme in the spermatid's tail and progressively differentiate. One of them becomes the major derivative with paracrystalline material accumulation, while the other one loses its volume and becomes the minor mitochondrial derivative. As part of the spermatid maturation, the nuclei also elongate while their volume decrease as the chromatin structure condensates and histones are replaced by protamines. Individualization, the process which establishes the individual sperms starts with the formation of the individualization complex (IC), which contains the F-actin-rich cone-shaped investment cones that are forming around the elongated nuclei. The IC migrates from the nuclei towards the tail end of the cyst, during its progression the majority of the cytosol and its content are displaced. An individual membrane starts to develop around each spermatid and a cystic bulge emerges. A non-apoptotic caspase activity facilitates the process of individualization 4. The caspase activity is mainly restricted to the cystic bulges and later in the waste bags at the end of the individualization 4,5. Remodeling of the round spermatocytes to functioning sperms is requiring strict regulation, selective protein degradation, and sufficient proteasome activity 6. There are multiple ubiquitin ligases known to have a role in the different steps of spermatogenesis (e.g.: Parkin, Ntc/Cul1/SkpA-SCF Ntc) 7,8 , and testis-specific proteasome subunits are emphasizing the ubiquitin-proteasome system's (UPS) role 9. E1, E2, and E3 orchestrate the ubiquitination of proteins inducing their degradation. While there are only a few types of E1 and E2 enzymes, many E3 enzymes were identified 10,11. E3 ligases can mark proteins for time and tissue-specific degradation therefore they have a role in many different cellular processes such as cell cycle, epigenetic regulation, mitophagy, etc. 12. Two E3 ligase complexes are necessary to activate the non-apoptotic caspase
bioRxiv (Cold Spring Harbor Laboratory), Dec 12, 2022
ABSTRACTMucormycosis is an invasive fungal infection caused by certain members of the fungal orde... more ABSTRACTMucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the generaRhizopus, LichtheimiaandMucor. The frequency of systemic mucormycosis has been increasing, mainly because of the elevating numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein duringRhizopus delemar-induced mucormycosis. We identified 17cotH-likegenes in theMucor lusitanicusgenome database. Successful disruption of fivecotHgenes was performed using the CRISPR-Cas9 system inMucor. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and thus, the composition of the spore wall. Furthermore, we demonstrate that spore size is dependent on thecotH4gene. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and DKA mouse model.IMPORTANCECurrent treatment options for mucormycosis are inadequate resulting in high mortality rates especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis ofRhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence ofMucor lusitanicusby using three infection models. We also report on the importance of one of these proteins, CotH4, to the spore wall formation through affecting the chitin content. Therefore, our studies extend the importance of CotH proteins toMucorand identifies the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.
A biologia BSc zarovizsga tetelek rovid vazlatos osszefoglalasa, az osszefuggesek kiemelese az eg... more A biologia BSc zarovizsga tetelek rovid vazlatos osszefoglalasa, az osszefuggesek kiemelese az egyes temak kozt.
Frontiers in Cell and Developmental Biology, Sep 29, 2021
Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary cons... more Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubuleorganizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. Here we describe the existence of a novel, tissue-specific γ-TuRC in Drosophila. We characterize three genes encoding testis-specific components of γ-TuRC (t-γ-TuRC) and find that presence of t-γ-TuRC is essential to male fertility. We show the diverse subcellular distribution of the t-γ-TuRC proteins during postmeiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. We also prove the physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1. Our results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC.
Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, whi... more Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which ...
bioRxiv (Cold Spring Harbor Laboratory), Mar 20, 2024
The initiation of microtubule formation in eukaryotes is primarily facilitated by γ-tubulin and t... more The initiation of microtubule formation in eukaryotes is primarily facilitated by γ-tubulin and the highly conserved protein complex called γ-Tubulin Ring Complex (γ-TuRC) in various microtubule-organizing centers (MTOCs). Non-centrosomal MTOCs have a range of functions and contribute to diverse cellular activities in different cell types. While the heterogeneity of tissue-specific MTOCs and γ-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. In this study, we investigate the testis-specific distribution of the canonical γ-TuRC by in vivo tagging of two complex members, Grip163 and Grip84. We find that both the testis-specific (t-γ-TuRC) and canonical form of γ-TuRC are simultaneously present and colocalize with each other at the basal body, apical nuclear tip, and near the elongating mitochondrial derivatives in the post-meiotic spermatids. We propose the possibility of the formation of MTOCs containing both t-γ-TuRC and γ-TuRC complexes. We also find some of the known γ-TuRC binding partners localizing to the apical nuclear tip. Additionally, we identify the Golgi localization of Grip84 in spermatocytes, alongside its centrosomal and diverse post-meiotic localization. These results highlight and prove the importance of the different γ-TuRCs in organizing the diverse MTOCs during the extensive rearrangement of cell organelles in Drosophila spermatogenesis. .
International journal of molecular sciences, Apr 3, 2024
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Muc... more Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Advances in Clinical and Experimental Medicine, Aug 24, 2022
There are numerous surprising discoveries in current comprehensive biopolymer research, including... more There are numerous surprising discoveries in current comprehensive biopolymer research, including the description of new types of biopolymers and the extension of their applications. The discovery of a new rotiferspecific biopolymer family (Rotimers) and the exceptional ability of these micrometazoans to inactivate and catabolize human-type neurotoxic aggregates (e.g., beta-amyloids, alpha-synucleins, prions) by their exudates can be mentioned as the original work of our research group. Rotimers are exogenous and protein complex molecules with a calcium-dependent production mechanism in both bdelloid and monogonant rotifers. However, their experimental and application possibilities are still unknown; only part of the class has been explored and described. Current Rotimer-related studies present promising biodiversity and bioactivity of these biomaterials (e.g., anti-and disaggregation effects or high degrees of adhesion to other molecules). The primary objective of current research is to explore and develop their application in translational biomedicine. A key area is the design of drug candidates against neurodegeneration-related aggregates based on the molecular information provided by the composition, structure and function of Rotimers. These novel biomaterials have the potential to open new perspectives in the pharmaceutical industry and healthcare.
The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effec... more The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, we used a comprehensive set of Drosophila Rabs for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. We confirm over 25 interactions for Rab2,
Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. The... more Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrial derivatives in spermatids. Testis-specific genes are less conserved and could gain new functions, thus raising a question whether Bb8 has retained its original enzymatic activity. We show that while Bb8 displays glutamate dehydrogenase activity, there are significant functional differences between the housekeeping Gdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8ms mutant phenotype, with superior performance by GLUD2. We also tested the role of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of our study indicate that Droso...
Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking acti... more Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin.
Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria fr... more Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates-both from keratomycosis and environment-were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.
bioRxiv (Cold Spring Harbor Laboratory), May 31, 2023
Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, whi... more Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which result in similarly abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms and interrelationships of PP4 and Ctb in the regulation of HR repair.
Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogen... more Drosophila melanogaster sperm reach an extraordinary long size, 1.8 mm, by the end of spermatogenesis. The mitochondrial derivatives run along the entire flagellum and provide structural rigidity for flagellar movement, but its precise function and organization is incompletely understood. The two mitochondrial derivatives differentiate and by the end of spermatogenesis the minor one reduces its size and the major one accumulates paracrystalline material inside it. The molecular constituents and precise function of the paracrystalline material have not yet been revealed. Here we purified the paracrystalline material from mature sperm and identified by mass spectrometry Sperm-Leucylaminopeptidase (S-Lap) family members as important constituents of it. To study the function of SLap proteins we show the characterization of classical mutants and RNAi lines affecting of the SLap genes and the analysis of their mutant phenotypes. We show that the male sterile phenotype of the SLap mutants is caused by defects in paracrystalline material accumulation and abnormal structure of the elongated major mitochondrial derivatives. Our work shows that SLap proteins localize and accumulate in the paracrystalline material of the major mitochondrial derivative. Therefore, we propose that SLap proteins are important constituents of the paracrystalline material of Drosophila melanogaster sperm.
International Journal of Molecular Sciences, Jan 19, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelag... more The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology. Drosophila melanogaster testis is a particularly suitable model to follow the process of spermatogenesis 1,2. During Drosophila spermatogenesis from a single spermatogonia sixty-four sperms are being produced, through mitotic and meiotic cell divisions and intensive cellular remodeling. After each division, the daughter cells remain in connection with plasma bridges, which allows them to develop simultaneously in a syncytial cyst. The early transitamplifying mitotic divisions result in sixteen spermatocytes. Spermatocytes undergo a maturing process, they have high transcriptional activity until meiosis and the accumulated transcripts contribute to the development of the transcriptionally mostly inactive post-meiotic stages 3. After meiosis the mitochondria aggregate and fuse to establish the nebenkern structure, which consists of two mitochondrial derivatives. Next, the spermatids start to elongate to reach a full length of ~ 1.8 mm, which approximately 150 times longer than their initial diameter. The two mitochondrial derivatives run along the axoneme in the spermatid's tail and progressively differentiate. One of them becomes the major derivative with paracrystalline material accumulation, while the other one loses its volume and becomes the minor mitochondrial derivative. As part of the spermatid maturation, the nuclei also elongate while their volume decrease as the chromatin structure condensates and histones are replaced by protamines. Individualization, the process which establishes the individual sperms starts with the formation of the individualization complex (IC), which contains the F-actin-rich cone-shaped investment cones that are forming around the elongated nuclei. The IC migrates from the nuclei towards the tail end of the cyst, during its progression the majority of the cytosol and its content are displaced. An individual membrane starts to develop around each spermatid and a cystic bulge emerges. A non-apoptotic caspase activity facilitates the process of individualization 4. The caspase activity is mainly restricted to the cystic bulges and later in the waste bags at the end of the individualization 4,5. Remodeling of the round spermatocytes to functioning sperms is requiring strict regulation, selective protein degradation, and sufficient proteasome activity 6. There are multiple ubiquitin ligases known to have a role in the different steps of spermatogenesis (e.g.: Parkin, Ntc/Cul1/SkpA-SCF Ntc) 7,8 , and testis-specific proteasome subunits are emphasizing the ubiquitin-proteasome system's (UPS) role 9. E1, E2, and E3 orchestrate the ubiquitination of proteins inducing their degradation. While there are only a few types of E1 and E2 enzymes, many E3 enzymes were identified 10,11. E3 ligases can mark proteins for time and tissue-specific degradation therefore they have a role in many different cellular processes such as cell cycle, epigenetic regulation, mitophagy, etc. 12. Two E3 ligase complexes are necessary to activate the non-apoptotic caspase
bioRxiv (Cold Spring Harbor Laboratory), Dec 12, 2022
ABSTRACTMucormycosis is an invasive fungal infection caused by certain members of the fungal orde... more ABSTRACTMucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the generaRhizopus, LichtheimiaandMucor. The frequency of systemic mucormycosis has been increasing, mainly because of the elevating numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein duringRhizopus delemar-induced mucormycosis. We identified 17cotH-likegenes in theMucor lusitanicusgenome database. Successful disruption of fivecotHgenes was performed using the CRISPR-Cas9 system inMucor. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and thus, the composition of the spore wall. Furthermore, we demonstrate that spore size is dependent on thecotH4gene. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and DKA mouse model.IMPORTANCECurrent treatment options for mucormycosis are inadequate resulting in high mortality rates especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis ofRhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence ofMucor lusitanicusby using three infection models. We also report on the importance of one of these proteins, CotH4, to the spore wall formation through affecting the chitin content. Therefore, our studies extend the importance of CotH proteins toMucorand identifies the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.
A biologia BSc zarovizsga tetelek rovid vazlatos osszefoglalasa, az osszefuggesek kiemelese az eg... more A biologia BSc zarovizsga tetelek rovid vazlatos osszefoglalasa, az osszefuggesek kiemelese az egyes temak kozt.
Frontiers in Cell and Developmental Biology, Sep 29, 2021
Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary cons... more Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubuleorganizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. Here we describe the existence of a novel, tissue-specific γ-TuRC in Drosophila. We characterize three genes encoding testis-specific components of γ-TuRC (t-γ-TuRC) and find that presence of t-γ-TuRC is essential to male fertility. We show the diverse subcellular distribution of the t-γ-TuRC proteins during postmeiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. We also prove the physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1. Our results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC.
Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, whi... more Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating the cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells lacking PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which ...
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Papers by Rita Sinka