Biomedical subjects

Brian T Chait

Publications and source records attributed to Brian T Chait.

60 records · Page 4Linked to original sources

PubMed2002

The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2.

The corepressors N-CoR and SMRT partner with histone deacetylases (HDACs) in diverse repression pathways. We report here that GPS2, a protein involved in intracellular signaling, is an integral subunit of the N-CoR-HDAC3 complex. We have determined structural motifs that direct the formation of a highly stable and active deacetylase complex. GPS2 and TBL1, another component of the N-CoR-HDAC3 complex, interact cooperatively with repression domain 1 of N-CoR to form a heterotrimeric structure and are indirectly linked to HDAC3 via an extended N-CoR SANT domain that also activates latent HDAC3 activity. More importantly, we show here that the N-CoR-HDAC3 complex inhibits JNK activation through the associated GPS2 subunit and thus could potentially provide an alternative mechanism for hormone-mediated antagonism of AP-1 function.

Amino Acid Sequence↗

PubMed2002

ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.

The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

Abscisic Acid↗

PubMed2002

N-terminal contributions of the gamma-subunit of fetal hemoglobin to its tetramer strength: remote effects at subunit contacts.

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.

Amino Acid Sequence↗

PubMed2001

SYT associates with human SNF/SWI complexes and the C-terminal region of its fusion partner SSX1 targets histones.

A global transcriptional co-activator, the SNF/SWI complex, has been characterized as a chromatin remodeling factor that enhances accessibility of the transcriptional machinery to DNA within a repressive chromatin structure. On the other hand, mutations in some human SNF/SWI complex components have been linked to tumor formation. We show here that SYT, a partner protein generating the synovial sarcoma fusion protein SYT-SSX, associates with native human SNF/SWI complexes. The SYT protein has a unique QPGY domain, which is also present in the largest subunits, p250 and the newly identified homolog p250R, of the corresponding SNF/SWI complexes. The C-terminal region (amino acids 310-387) of SSX1, comprising the SSX1 portion of the SYT-SSX1 fusion protein, binds strongly to core histones and oligonucleosomes in vitro and directs nuclear localization of a green fluorescence protein fusion protein. Experiments with serial C-terminal deletion mutants of SSX1 indicate that these properties map to a common region and also correlate with the previously demonstrated anchorage-independent colony formation activity of SYT-SSX in Rat 3Y1 cells. These data suggest that SYT-SSX interferes with the function of either the SNF/SWI complexes or another SYT-interacting co-activator, p300, by changing their targeted localization or by directly inhibiting their chromatin remodeling activities.

Amino Acid Sequence↗

PubMedDate not supplied

A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes.

A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form. Here we describe a peptide, Bio-Ox, that mimics the immunoglobulin G (IgG) binding interface of Staphylococcus aureus Protein A, and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose. We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins, and can be applied to a variety of structural genomics and proteomics studies.

Bacterial Proteins↗

PubMedDate not supplied

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Amino Acid Sequence↗