Lourdes M. Aleman - Academia.edu (original) (raw)

Papers by Lourdes M. Aleman

Journal of Biological Chemistry, Nov 1, 2000

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus em... more We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. Nck is an Src homology (SH) 1 2/SH3 adaptor protein, which

Biochemistry, Sep 25, 2002

Protein-protein interactions play a critical role in cellular processes such as signal transducti... more Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Oncogene, Jul 5, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

Molecular Microbiology, Sep 16, 2010

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced ... more The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5Ј-and 3Ј-ends of 16S rRNA as well as maturation of the 5Ј-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Exploring the specificity and mechanisms of siRNA-mediated gene silencing in mammalian cells

Proceedings of the National Academy of Sciences, 2000

Two distinct benzodiazepine binding sites have been identified, ( i ) a central site restricted t... more Two distinct benzodiazepine binding sites have been identified, ( i ) a central site restricted to brain and ( ii ) a ubiquitously expressed mitochondrial binding site, the so-called peripheral-type benzodiazepine receptor (PBR). In this paper, we show that a benzazepine referred to as BBL22 (2-amino 9-chloro-7-(2-fluorophenyl)-5H-pyrimidol[5,4- d ][2]benzazepine), which is classified as a PBR ligand based on structure, induces arrest in G 2 /M phase of the cell cycle in human tumor cell lines of both epithelial and hematopoietic cellular origin. After G 2 /M arrest, several tumor types, notably prostate and certain breast cancer lines exhibited significant apoptosis. Ideally, cancer therapies should selectively target tumor cells while sparing normal cell counterparts. BBL22 exhibited such selectivity, as it did not affect the growth and survival of nonmalignant breast and prostate epithelial lines. Moreover, BBL22 demonstrated structural requirements for this selective antitumor a...

Molecular Microbiology, 2010

SummaryThe UPF0054 protein family is highly conserved with homologues present in nearly every seq... more SummaryThe UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′‐ and 3′‐ends of 16S rRNA as well as maturation of the 5′‐termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of high...

Journal of Biological Chemistry, 2000

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus em... more We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. Nck is an Src homology (SH) 1 2/SH3 adaptor protein, which

International Journal of Intelligent Defence Support Systems, 2010

ABSTRACT Teaching about proteins, how their structure is specified, and how in turn this influenc... more ABSTRACT Teaching about proteins, how their structure is specified, and how in turn this influences their function has become an integral curriculum component for both introductory biology and biochemistry undergraduate courses. To understand how proteins work, students first learn about protein structure and its relationship to protein function. StarBiochem is a protein visualization tool that is used to assist in teaching key structural biology concepts in an interactive manner. StarBiochem allows for the visualization and manipulation of PBD (Protein Data Bank) molecules in a 3D environment. In this manuscript, we provide examples of StarBiochem usage within high school and undergraduate curricula. Specifically, we will highlight how StarBiochem is currently used in undergraduate courses at the Massachusetts Institute of Technology (MIT), Brandeis University, and in high school outreach programs at the Broad Institute of Harvard and MIT, and at the MIT Museum.

Biochemistry, 2002

Protein-protein interactions play a critical role in cellular processes such as signal transducti... more Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. si... more Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. siRNAs bind to their target sequence and guide transcript cleavage and subsequent degradation. This type of silencing is associated with equivalent levels of RNA and protein knockdown. siRNA-mediated knockdown was originally thought to be highly specific. However, the downregulation of non-target mRNAs has been observed following transfection of siRNAs in human cells. Many of these RNA changes are due to siRNA binding to partially complementary sequences within nontargeted transcripts and therefore are termed "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing a variety of base pairing interactions in the 9 th , 10 th , and 11 th positions of two siRNA binding sites located in a reporter gene. This region was chosen because siRNA-mediated transcript cleavage occurs between the 10 th and 11 th positions of the mRNA:siRNA duplex. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 resulted in a twofold or more mRNA decrease, with varying degrees of protein knockdown. mRNA and protein analysis revealed combinations for which the resulting mRNA and protein levels did not correlate. Although siRNA-mediated transcript cleavage is catalyzed by the endonuclease Argonaute 2 (Ago2), knockdown of Ago2 expression did not affect mRNA knockdown for imperfectly complementary combinations. These results indicate that off-target mRNA reductions are likely attributable to Ago2-independent degradation processes. Using the same reporter system we have also uncovered instances in which complementary siRNAs resulted in high protein/RNA knockdown ratios with dramatic protein silencing. For these particular combinations, the disparity between RNA and protein knockdown is dependent on Ago2 function. This may suggest that the sequences within atypical complementary mRNA:siRNA combinations result in unproductive cleavage by Ago2, leading to persistent binding and enhanced silencing through translational repression. Our findings demonstrate that differences within complementary target sequences can lead to differences in the type of silencing mechanisms that result. The results presented in this dissertation also provide a better understanding of how off-target effects mediate mRNA and protein knockdown of unrelated transcripts, with meaningful implications for those using siRNAs as a tool.

RNA, 2007

The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray r... more The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered “off-target” effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3′ UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9–11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resultin...

Oncogene, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

…, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

Journal of Biological Chemistry, Nov 1, 2000

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus em... more We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. Nck is an Src homology (SH) 1 2/SH3 adaptor protein, which

Biochemistry, Sep 25, 2002

Protein-protein interactions play a critical role in cellular processes such as signal transducti... more Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Oncogene, Jul 5, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

Molecular Microbiology, Sep 16, 2010

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced ... more The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5Ј-and 3Ј-ends of 16S rRNA as well as maturation of the 5Ј-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Exploring the specificity and mechanisms of siRNA-mediated gene silencing in mammalian cells

Proceedings of the National Academy of Sciences, 2000

Two distinct benzodiazepine binding sites have been identified, ( i ) a central site restricted t... more Two distinct benzodiazepine binding sites have been identified, ( i ) a central site restricted to brain and ( ii ) a ubiquitously expressed mitochondrial binding site, the so-called peripheral-type benzodiazepine receptor (PBR). In this paper, we show that a benzazepine referred to as BBL22 (2-amino 9-chloro-7-(2-fluorophenyl)-5H-pyrimidol[5,4- d ][2]benzazepine), which is classified as a PBR ligand based on structure, induces arrest in G 2 /M phase of the cell cycle in human tumor cell lines of both epithelial and hematopoietic cellular origin. After G 2 /M arrest, several tumor types, notably prostate and certain breast cancer lines exhibited significant apoptosis. Ideally, cancer therapies should selectively target tumor cells while sparing normal cell counterparts. BBL22 exhibited such selectivity, as it did not affect the growth and survival of nonmalignant breast and prostate epithelial lines. Moreover, BBL22 demonstrated structural requirements for this selective antitumor a...

Molecular Microbiology, 2010

SummaryThe UPF0054 protein family is highly conserved with homologues present in nearly every seq... more SummaryThe UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′‐ and 3′‐ends of 16S rRNA as well as maturation of the 5′‐termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of high...

Journal of Biological Chemistry, 2000

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus em... more We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo. Nck is an Src homology (SH) 1 2/SH3 adaptor protein, which

International Journal of Intelligent Defence Support Systems, 2010

ABSTRACT Teaching about proteins, how their structure is specified, and how in turn this influenc... more ABSTRACT Teaching about proteins, how their structure is specified, and how in turn this influences their function has become an integral curriculum component for both introductory biology and biochemistry undergraduate courses. To understand how proteins work, students first learn about protein structure and its relationship to protein function. StarBiochem is a protein visualization tool that is used to assist in teaching key structural biology concepts in an interactive manner. StarBiochem allows for the visualization and manipulation of PBD (Protein Data Bank) molecules in a 3D environment. In this manuscript, we provide examples of StarBiochem usage within high school and undergraduate curricula. Specifically, we will highlight how StarBiochem is currently used in undergraduate courses at the Massachusetts Institute of Technology (MIT), Brandeis University, and in high school outreach programs at the Broad Institute of Harvard and MIT, and at the MIT Museum.

Biochemistry, 2002

Protein-protein interactions play a critical role in cellular processes such as signal transducti... more Protein-protein interactions play a critical role in cellular processes such as signal transduction. Although many methods for identifying the binding partners of a protein of interest are available, it is currently difficult or impossible to assess the functional consequences of a specific interaction in vivo. To address this issue, we propose to modify proteins by addition of an artificial protein binding interface, thereby forcing them to interact in the cell in a pairwise fashion and allowing the functional consequences to be determined. For this purpose, we have developed an artificial binding interface consisting of a anti-Myc single-chain antibody (ScFv) and its peptide epitope. We found that the binding of an ScFv derived from anti-Myc monoclonal antibody 9E10 was relatively weak in vivo, so we selected an improved clone, 3DX, by in vitro mutagenesis and phage display. 3DX bound well to its epitope in a yeast two-hybrid system, and GST-fused 3DX also bound to several Myc-tagged proteins in mammalian cells. In vivo binding was relatively insensitive to the position of the ScFv in a fusion protein, but was improved by including multiple tandem copies of the Myc epitope in the binding partner. To test the system, we successfully replaced the SH3 domain-mediated interaction between the Abl tyrosine kinase and adaptor proteins Crk and Nck with an engineered interaction between 3DX and multiple Myc tags. We expect that this approach, which we term a functional interaction trap, will be a powerful proteomic tool for investigating protein-protein interactions.

Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. si... more Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. siRNAs bind to their target sequence and guide transcript cleavage and subsequent degradation. This type of silencing is associated with equivalent levels of RNA and protein knockdown. siRNA-mediated knockdown was originally thought to be highly specific. However, the downregulation of non-target mRNAs has been observed following transfection of siRNAs in human cells. Many of these RNA changes are due to siRNA binding to partially complementary sequences within nontargeted transcripts and therefore are termed "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing a variety of base pairing interactions in the 9 th , 10 th , and 11 th positions of two siRNA binding sites located in a reporter gene. This region was chosen because siRNA-mediated transcript cleavage occurs between the 10 th and 11 th positions of the mRNA:siRNA duplex. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 resulted in a twofold or more mRNA decrease, with varying degrees of protein knockdown. mRNA and protein analysis revealed combinations for which the resulting mRNA and protein levels did not correlate. Although siRNA-mediated transcript cleavage is catalyzed by the endonuclease Argonaute 2 (Ago2), knockdown of Ago2 expression did not affect mRNA knockdown for imperfectly complementary combinations. These results indicate that off-target mRNA reductions are likely attributable to Ago2-independent degradation processes. Using the same reporter system we have also uncovered instances in which complementary siRNAs resulted in high protein/RNA knockdown ratios with dramatic protein silencing. For these particular combinations, the disparity between RNA and protein knockdown is dependent on Ago2 function. This may suggest that the sequences within atypical complementary mRNA:siRNA combinations result in unproductive cleavage by Ago2, leading to persistent binding and enhanced silencing through translational repression. Our findings demonstrate that differences within complementary target sequences can lead to differences in the type of silencing mechanisms that result. The results presented in this dissertation also provide a better understanding of how off-target effects mediate mRNA and protein knockdown of unrelated transcripts, with meaningful implications for those using siRNAs as a tool.

RNA, 2007

The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray r... more The downregulation of many mRNAs has been observed through bioinformatic analysis of microarray results following transfection of short interfering RNAs (siRNAs). Many of these mRNA changes are due to the interaction of the siRNA guide strand with partially complementary sites and thus are considered “off-target” effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing single and double mismatches, bulges, and noncanonical base-pairing interactions in the 9th, 10th, and 11th positions of two siRNA binding sites located in the 3′ UTR of an integrated reporter gene. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9–11 result in a twofold or more mRNA decrease with varying degrees of protein knockdown. However, mRNA and protein analysis of the various mRNA:siRNA combinations reveals instances in which mRNA and protein levels do not correlate. Analysis of the resultin...

Oncogene, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

…, 2001

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimul... more The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate eectors for Nck function. Numerous additional SH2-and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We ®nd that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Ecient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.