Aftabul Haque | Massachusetts Institute of Technology (MIT) (original) (raw)

Papers by Aftabul Haque

Journal of Biological Chemistry, 2013

Background: PTP␣ has been implicated in breast cancer, but its function remains to be defined. Re... more Background: PTP␣ has been implicated in breast cancer, but its function remains to be defined. Results: Suppression of PTP␣ led to a GRB7-dependent, ErbB2-mediated increase in mammary epithelial cell migration. PTP␣ dephosphorylated FAK on Tyr-407. Conclusion: PTP␣ functions to suppress ErbB2 signaling events that lead to migration of breast cancer cells. Significance: PTP␣ may play positive or negative roles in signaling, depending upon context.

Cell, 2011

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and le... more Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the b subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

Communications biology, Oct 30, 2022

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Iden... more Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including l... more The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including late-onset Alzheimer's disease (AD). We investigate the cellular consequences of APOE4 in human iPSC-derived astrocytes, observing an endocytic defect in APOE4 astrocytes compared with their isogenic APOE3 counterparts. Given the evolutionarily conserved nature of endocytosis, we built a yeast model to identify genetic modifiers of the endocytic defect associated with APOE4. In yeast, only the expression of APOE4 results in dose-dependent defects in both endocytosis and growth. We discover that increasing expression of the early endocytic adaptor protein Yap1802p, a homolog of the human AD risk factor PICALM, rescues the APOE4-induced endocytic defect. In iPSC-derived human astrocytes, increasing expression of PICALM similarly reverses endocytic disruptions. Our work identifies a functional interaction between two AD genetic risk factors-APOE4 and PICALM-centered on the conserved biol...

Frontiers in Immunology

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is f... more Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a Kd of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a Kd for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a Kd of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a Kd of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a Kd estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a Kd of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 (Kd 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.

Molecular cell, 2018

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipi... more In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD...

Cell, 2011

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and le... more Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the β subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

The FEBS journal, 2014

The transient inactivation of protein phosphatases contributes to the efficiency and temporal con... more The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of colocalized NADPH oxidases and is mediated by locally produced reactive oxygen species, in particular H2 O2 . How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of thioredoxin 1(TRX1) slows PTEN reactivation in intact living cells. Finally, using a mechanism-based trapping approach, we demonstrate ...

Nature Protocols, 2012

We describe a phage display approach that we have previously used to generate conformation-sensor... more We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that specifically recognize and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (ptp1B). We use a solutionbased panning and screening strategy conducted in the presence of reduced active ptp1B, which enriches antibodies to epitopes unique to the oxidized form while excluding antibodies that recognize epitopes common to oxidized and reduced forms of ptp1B. this strategy avoids conventional solid-phase immobilization owing to its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects single-chain variable fragments (scFvs) directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. these conformation-specific scFvs illustrate that stabilization of oxidized ptp1B is an effective strategy to inhibit ptp1B function; it is possible that this approach may be applicable to the protein tyrosine phosphatase (ptp) family as a whole. With this protocol, isolation and characterization of specific scFvs from immune responsive animals should take ~6 weeks.

We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ... more We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a β1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new...

Journal of Immunological Methods, 2004

Exploitation of recombinant technology to study proteins containing strings of short consensus re... more Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.

Journal of Biological Chemistry, 2013

Background: PTP␣ has been implicated in breast cancer, but its function remains to be defined. Re... more Background: PTP␣ has been implicated in breast cancer, but its function remains to be defined. Results: Suppression of PTP␣ led to a GRB7-dependent, ErbB2-mediated increase in mammary epithelial cell migration. PTP␣ dephosphorylated FAK on Tyr-407. Conclusion: PTP␣ functions to suppress ErbB2 signaling events that lead to migration of breast cancer cells. Significance: PTP␣ may play positive or negative roles in signaling, depending upon context.

Cell, 2011

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and le... more Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the b subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

Communications biology, Oct 30, 2022

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Iden... more Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including l... more The ε4 allele of apolipoprotein E (APOE4) is a genetic risk factor for many diseases, including late-onset Alzheimer's disease (AD). We investigate the cellular consequences of APOE4 in human iPSC-derived astrocytes, observing an endocytic defect in APOE4 astrocytes compared with their isogenic APOE3 counterparts. Given the evolutionarily conserved nature of endocytosis, we built a yeast model to identify genetic modifiers of the endocytic defect associated with APOE4. In yeast, only the expression of APOE4 results in dose-dependent defects in both endocytosis and growth. We discover that increasing expression of the early endocytic adaptor protein Yap1802p, a homolog of the human AD risk factor PICALM, rescues the APOE4-induced endocytic defect. In iPSC-derived human astrocytes, increasing expression of PICALM similarly reverses endocytic disruptions. Our work identifies a functional interaction between two AD genetic risk factors-APOE4 and PICALM-centered on the conserved biol...

Frontiers in Immunology

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is f... more Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a Kd of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a Kd for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a Kd of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a Kd of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a Kd estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a Kd of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 (Kd 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.

Molecular cell, 2018

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipi... more In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD...

Cell, 2011

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and le... more Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the β subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.

The FEBS journal, 2014

The transient inactivation of protein phosphatases contributes to the efficiency and temporal con... more The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of colocalized NADPH oxidases and is mediated by locally produced reactive oxygen species, in particular H2 O2 . How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of thioredoxin 1(TRX1) slows PTEN reactivation in intact living cells. Finally, using a mechanism-based trapping approach, we demonstrate ...

Nature Protocols, 2012

We describe a phage display approach that we have previously used to generate conformation-sensor... more We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that specifically recognize and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (ptp1B). We use a solutionbased panning and screening strategy conducted in the presence of reduced active ptp1B, which enriches antibodies to epitopes unique to the oxidized form while excluding antibodies that recognize epitopes common to oxidized and reduced forms of ptp1B. this strategy avoids conventional solid-phase immobilization owing to its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects single-chain variable fragments (scFvs) directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. these conformation-specific scFvs illustrate that stabilization of oxidized ptp1B is an effective strategy to inhibit ptp1B function; it is possible that this approach may be applicable to the protein tyrosine phosphatase (ptp) family as a whole. With this protocol, isolation and characterization of specific scFvs from immune responsive animals should take ~6 weeks.

We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ... more We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a β1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new...

Journal of Immunological Methods, 2004

Exploitation of recombinant technology to study proteins containing strings of short consensus re... more Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.