Michał Milewski - Academia.edu (original) (raw)
Papers by Michał Milewski
Cellular & Molecular Biology Letters, 2002
Genes, 2021
Congenital microcephaly causes smaller than average head circumference relative to age, sex and e... more Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confi...
Genes, 2021
Congenital microcephaly causes smaller than average head circumference relative to age, sex and e... more Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confi...
disease-causing protein. The most promising approaches include (1) the development of specific an... more disease-causing protein. The most promising approaches include (1) the development of specific antibodies or small peptides that bind the aggregating proteins and reduce their aggregation level, (2) using molecular chaperones to increase the efficiency of proper protein folding, and thus reduce the aggregation rate, and finally (3) the development of the RNAi-based therapy, aimed at silencing the genes encoding the aggregating proteins. Acknowledgements: Supported by grants PBZ/KBN/122/P05/01-08, PBZ/KBN/122/
Journal of Biological Chemistry, 2000
Polarization of CFTR, a cAMP-activated Cl channel to the apical plasma membrane in epithelial cel... more Polarization of CFTR, a cAMP-activated Cl channel to the apical plasma membrane in epithelial cells is critical for vectorial Cl transport. Previously, we reported that the C-terminus of CFTR constitutes a PDZ interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial Cl secretion. Point substitution of the C-terminal leucine (L at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and transepithelial Cl secretion. Point substitution of the threonine (T at position-2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as transepithelial Cl secretion. By contrast, individual point substitution of the other C-terminal amino acids (Q at position-4, D at position-3 and R at position-1) with alanine had no effect on measured parameters. We conclude that the PDZ interacting domain, in particular the leucine (position 0) and threonine (position-2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate transepithelial Cl secretion. Our data suggest that mutations that delete the C-terminus of CFTR may cause cystic fibrosis in part because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.
on-line at: www.actabp.pl Phenotype modifiers of spinal muscular atrophy: the number of SMN2 gene... more on-line at: www.actabp.pl Phenotype modifiers of spinal muscular atrophy: the number of SMN2 gene copies, deletion in the NAIP gene and probably gender influence the course of the disease
Postepy biochemii, 2015
Torsin 1A is a protein mutated in torsion dystonia type 1, a hereditary neurological disorder of ... more Torsin 1A is a protein mutated in torsion dystonia type 1, a hereditary neurological disorder of early onset and variable clinical picture. The basic cellular function of torsin 1A, a polypeptide localized predominantly in the endoplasmic reticulum and nuclear envelope, remains unknown, although the protein is suspected of being involved in many different cellular processes, including regulating a proper structure and function of nuclear envelope, contributing to the synaptic vesicular trafficking, or assisting in proper folding of misfolded proteins. This review summarizes the current state of knowledge regarding the potential functions of torsin 1A in the context of hypothetical pathomechanisms responsible for torsion dystonia type 1.
Postepy biochemii, 2015
Huntington's disease is a progressive neurodegenerative disorder of genetic origin that still... more Huntington's disease is a progressive neurodegenerative disorder of genetic origin that still lacks an effective treatment. Recently, a number of new attempts have been undertaken to develop a successful molecular therapy for this incurable condition. The novel approaches employ, among others, some new methods to selectively silence the mutated gene or to neutralize its toxic protein product. This paper reviews all major strategies that are currently considered for molecular therapy of Huntington's disease while discussing their potential effectiveness regarding the treatment of both the Huntington's disease and a large group of related neurodegenerative disorders associated with abnormal protein aggregation.
Postepy biochemii, 2005
The so-called conformational diseases constitute a specific subtype of protein folding diseases t... more The so-called conformational diseases constitute a specific subtype of protein folding diseases that is characterized by abnormal aggregation of improperly folded polypeptides. This review describes a series of examples of such disorders and summarizes the present knowledge on their molecular pathophysiology and new therapeutic strategies.
Introduction Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Pre... more Introduction Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Previous reports have suggested that torsin 1A, a protein mutated in this disease, might function as a chaperone that prevents the toxic aggregation of misfolded polypeptides. The aim of the study The aim of this study was to verify the chaperone function of torsin 1A by investigating its ability to prevent the aggregation of huntingtin model peptides. Materials and methods N-terminal mutant huntingtin fragments of different length were co-expressed in neuronal HT-22 and non-neuronal HeLa cells with either the wild-type or mutant (ΔE302/303) torsin 1A protein. The transfected cells were immunostained and analyzed for the presence of huntingtin aggregates using fluorescence microscopy. Results The immunofluorescence analysis of huntingtin subcellular distribution within the transfected cells showed no significant difference between the huntingtin aggregation levels in cells co-expressing the...
The article summarizes over 20 years of experience of a reference lab in fragile X mental retarda... more The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5′UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of F...
American journal of physiology. Lung cellular and molecular physiology, Dec 1, 2016
The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy fo... more The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Delet...
Genes, 2016
The article summarizes over 20 years of experience of a reference lab in fragile X mental retarda... more The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5 UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used.
Cell Biology International, Sep 1, 2010
The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptid... more The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so-called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co-aggregation properties of the N-terminal fragment of huntingtin fused with unrelated non-aggregating and/or self-aggregating peptides. We show that the ability of the mutated N-terminal huntingtin fragment to sequester non-related proteins can be significantly increased by fusion with the non-aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self-aggregating C-terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non-fused huntingtin fragments. We also demonstrate that the co-aggregation of different non-fused N-terminal huntingtin fragments depends on their length, with long fragments of the wild-type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co-aggregate with other peptides.
Ashrae Transactions, 2007
Cellular & Molecular Biology Letters, 2002
Genes, 2021
Congenital microcephaly causes smaller than average head circumference relative to age, sex and e... more Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confi...
Genes, 2021
Congenital microcephaly causes smaller than average head circumference relative to age, sex and e... more Congenital microcephaly causes smaller than average head circumference relative to age, sex and ethnicity and is most usually associated with a variety of neurodevelopmental disorders. The underlying etiology is highly heterogeneous and can be either environmental or genetic. Disruption of any one of multiple biological processes, such as those underlying neurogenesis, cell cycle and division, DNA repair or transcription regulation, can result in microcephaly. This etiological heterogeneity manifests in a clinical variability and presents a major diagnostic and therapeutic challenge, leaving an unacceptably large proportion of over half of microcephaly patients without molecular diagnosis. To elucidate the clinical and genetic landscapes of congenital microcephaly, we sequenced the exomes of 191 clinically diagnosed patients with microcephaly as one of the features. We established a molecular basis for microcephaly in 71 patients (37%), and detected novel variants in five high confi...
disease-causing protein. The most promising approaches include (1) the development of specific an... more disease-causing protein. The most promising approaches include (1) the development of specific antibodies or small peptides that bind the aggregating proteins and reduce their aggregation level, (2) using molecular chaperones to increase the efficiency of proper protein folding, and thus reduce the aggregation rate, and finally (3) the development of the RNAi-based therapy, aimed at silencing the genes encoding the aggregating proteins. Acknowledgements: Supported by grants PBZ/KBN/122/P05/01-08, PBZ/KBN/122/
Journal of Biological Chemistry, 2000
Polarization of CFTR, a cAMP-activated Cl channel to the apical plasma membrane in epithelial cel... more Polarization of CFTR, a cAMP-activated Cl channel to the apical plasma membrane in epithelial cells is critical for vectorial Cl transport. Previously, we reported that the C-terminus of CFTR constitutes a PDZ interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial Cl secretion. Point substitution of the C-terminal leucine (L at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and transepithelial Cl secretion. Point substitution of the threonine (T at position-2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as transepithelial Cl secretion. By contrast, individual point substitution of the other C-terminal amino acids (Q at position-4, D at position-3 and R at position-1) with alanine had no effect on measured parameters. We conclude that the PDZ interacting domain, in particular the leucine (position 0) and threonine (position-2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate transepithelial Cl secretion. Our data suggest that mutations that delete the C-terminus of CFTR may cause cystic fibrosis in part because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.
on-line at: www.actabp.pl Phenotype modifiers of spinal muscular atrophy: the number of SMN2 gene... more on-line at: www.actabp.pl Phenotype modifiers of spinal muscular atrophy: the number of SMN2 gene copies, deletion in the NAIP gene and probably gender influence the course of the disease
Postepy biochemii, 2015
Torsin 1A is a protein mutated in torsion dystonia type 1, a hereditary neurological disorder of ... more Torsin 1A is a protein mutated in torsion dystonia type 1, a hereditary neurological disorder of early onset and variable clinical picture. The basic cellular function of torsin 1A, a polypeptide localized predominantly in the endoplasmic reticulum and nuclear envelope, remains unknown, although the protein is suspected of being involved in many different cellular processes, including regulating a proper structure and function of nuclear envelope, contributing to the synaptic vesicular trafficking, or assisting in proper folding of misfolded proteins. This review summarizes the current state of knowledge regarding the potential functions of torsin 1A in the context of hypothetical pathomechanisms responsible for torsion dystonia type 1.
Postepy biochemii, 2015
Huntington's disease is a progressive neurodegenerative disorder of genetic origin that still... more Huntington's disease is a progressive neurodegenerative disorder of genetic origin that still lacks an effective treatment. Recently, a number of new attempts have been undertaken to develop a successful molecular therapy for this incurable condition. The novel approaches employ, among others, some new methods to selectively silence the mutated gene or to neutralize its toxic protein product. This paper reviews all major strategies that are currently considered for molecular therapy of Huntington's disease while discussing their potential effectiveness regarding the treatment of both the Huntington's disease and a large group of related neurodegenerative disorders associated with abnormal protein aggregation.
Postepy biochemii, 2005
The so-called conformational diseases constitute a specific subtype of protein folding diseases t... more The so-called conformational diseases constitute a specific subtype of protein folding diseases that is characterized by abnormal aggregation of improperly folded polypeptides. This review describes a series of examples of such disorders and summarizes the present knowledge on their molecular pathophysiology and new therapeutic strategies.
Introduction Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Pre... more Introduction Torsion dystonia type 1 is the most common form of early-onset primary dystonia. Previous reports have suggested that torsin 1A, a protein mutated in this disease, might function as a chaperone that prevents the toxic aggregation of misfolded polypeptides. The aim of the study The aim of this study was to verify the chaperone function of torsin 1A by investigating its ability to prevent the aggregation of huntingtin model peptides. Materials and methods N-terminal mutant huntingtin fragments of different length were co-expressed in neuronal HT-22 and non-neuronal HeLa cells with either the wild-type or mutant (ΔE302/303) torsin 1A protein. The transfected cells were immunostained and analyzed for the presence of huntingtin aggregates using fluorescence microscopy. Results The immunofluorescence analysis of huntingtin subcellular distribution within the transfected cells showed no significant difference between the huntingtin aggregation levels in cells co-expressing the...
The article summarizes over 20 years of experience of a reference lab in fragile X mental retarda... more The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5′UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of F...
American journal of physiology. Lung cellular and molecular physiology, Dec 1, 2016
The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy fo... more The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Delet...
Genes, 2016
The article summarizes over 20 years of experience of a reference lab in fragile X mental retarda... more The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5 UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used.
Cell Biology International, Sep 1, 2010
The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptid... more The sequestration of crucial cellular proteins into insoluble aggregates formed by the polypeptides containing expanded polyglutamine tracts has been proposed to be the key mechanism responsible for the abnormal cell functioning in the so-called polyglutamine diseases. To evaluate to what extent the ability of polyglutamine sequences to recruit other proteins into the intracellular aggregates depends on the composition of the aggregating peptide, we analysed the co-aggregation properties of the N-terminal fragment of huntingtin fused with unrelated non-aggregating and/or self-aggregating peptides. We show that the ability of the mutated N-terminal huntingtin fragment to sequester non-related proteins can be significantly increased by fusion with the non-aggregating reporter protein [GFP (green fluorescence protein)]. By contrast, fusion with the self-aggregating C-terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) dramatically reduces the sequestration of related non-fused huntingtin fragments. We also demonstrate that the co-aggregation of different non-fused N-terminal huntingtin fragments depends on their length, with long fragments of the wild-type huntingtin not only excluded from the nuclear inclusions, but also very inefficiently sequestered into the cytoplasmic aggregates formed by the short fragments of mutant protein. Additionally, our results suggest that atypical intracellular aggregation patterns, which include unusual distribution and/or morphology of protein aggregates, are associated with altered ability of accumulating proteins to co-aggregate with other peptides.
Ashrae Transactions, 2007