Biology subjects

Kempf, C.

Publications and source records attributed to Kempf, C..

5 recordsLinked to original sources

bioRxiv2026

Multicomplex Integrative Structural Modeling of a Human Histone Deacetylase Interactome

Histone Deacetylase (HDAC) 1 and 2 are key enzymatic components in multiple large chromatin remodeling complexes including NuRD, SIN3, and CoREST. In addition, both HDAC 1 and 2 contain a large intrinsically disordered region (IDR) within their C-terminal domain (CTD). How HDAC1/2 assemble into these complexes and the structure of the CTD IDR remains poorly understood. Here, we used HDAC1/2 to isolate their protein interaction networks from cells and used crosslinking mass spectrometry (XL-MS) coupled with the Integrative Modeling Platform to build structural models of the NuRD, SIN3A, and CoREST complexes. Next, we implemented an AlphaFold-enabled XL-MS constrained modeling approach to investigate how HDAC1 could assemble into these complexes. We show that the CTD IDR of HDAC1 folds into alpha helices in these complexes. Finally, we built a complete integrative structural model of a NuRD subcomplex including the abundant HDAC1:MBD3:MTA1:GATAD2B:RBBP4 subunits, which included 6 IDRs. The approaches used herein are broadly applicable for the study of protein complexes and protein interaction networks that can provide important insights into IDRs.

systems biology↗

bioRxiv2025

Integrative Structural Modeling of Intrinsically Disordered Regions in a Human HDAC2 Chromatin Remodeling Complex

Intrinsically disordered regions (IDRs) and intrinsically disordered proteins (IDPs) play pivotal roles in cellular signaling, molecular recognition, and the regulation of various biological processes. These flexible and conformationally dynamic protein segments are difficult to study using structural analysis methods and computational approaches including AlphaFold. Therefore a critical challenge arises when attempting to understand the structural basis of protein-protein interactions involving IDRs. Here we demonstrate that the poorly characterized C16orf87 protein, which we rename as MHAP1, forms a stable complex with HDAC2 and MIER1. These three proteins all contain IDRs whose structure is unknown. We implemented an integrative approach combining experimental crosslinking data with computational modeling techniques (I-TASSER, HADDOCK, AlphaFold) to probe the IDR-driven assembly of the HDAC1:MIER2:MHAP1 complex and build an integrative structural model of this complex. The C-terminal domain of HDAC2, a poorly characterized IDR, promotes interactions between the ELM2 domain of MIER1 as well as the N- and C-termini of MHAP1. These results contrast with most current literature, including the results from AlphaFold alone that are missing structural information on HDAC C-domain. The approach herein can be generalized to study other complexes, emphasizing the need for integrative approaches in determining the 3D structures of IDR/IDP-driven complexes.

biochemistry↗

bioRxiv2020

The impact of intravenous dodecafluoropentane on a murine model of acute lung injury

Acute hypoxemic respiratory failure presents therapeutic challenges due to ventilation/perfusion mismatch and shunt. The goal of management strategies is to improve arterial oxygenation, however each management strategy presents risk to patients from iatrogenic injury. Intravenous oxygen therapeutics present an appealing option to improve arterial oxygenation without these risks. We used a two-hit murine model of acute lung injury to evaluate the effect of intravenous dodecafluoropentane (NanO2) on oxygen saturation and bronchoalveolar lavage cell count and protein. Mice were given intratracheal lipopolysaccharide and 20 hours later were intubated and ventilated with high tidal volumes. NanO2 was given by bolus injection at the initiation of mechanical ventilation and again at 2 hours, while oxygen saturation was measured every 15 minutes. At the conclusion of the experiment (4 hours), a bronchoalveolar lavage was performed. There was no difference in mean O2 saturation at time zero, however the difference between the mean O2 saturation immediately prior to injection and the mean first O2 saturation after injection in the control saline group were 91% and 83%, mean difference -7.5%; whereas mean O2 saturation in the NanO2 treated group rose from 89% to 91%, mean difference +2.5%, net difference 10% [95% CI: 2.7,17.3], p=0.01). There was a statistically significant difference in cell count, but not protein, on the bronchoalveolar lavage analysis. These data show that NanO2 rapidly improves oxygen saturation in a two-hit model of acute lung injury, and shows potential as an intravenous oxygen therapeutic in the management in acute hypoxemic respiratory failure.

pharmacology and toxicology↗

bioRxiv2019

Differential complex formation via paralogs in the human Sin3 protein interaction network.

Despite the continued analysis of HDAC inhibitor efficacy in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The functional attributes of these protein complexes remain poorly characterized in humans. Contributing to the poor definition of Sin3 complex attributes in higher eukaryotes is the presence of two Sin3 scaffolding proteins, SIN3A and SIN3B. Here we show that paralog switching influences the interaction networks of the Sin3 complexes. While SIN3A and SIN3B do have unique interaction network components, we find that SIN3A and SIN3B interact with a common set of proteins. Additionally, our results suggest that SIN3A and SIN3B may possess the capacity to form hetero-oligomeric complexes. While one principal form of SIN3B exists in humans, the analysis of rare SIN3B proteoforms provides insight into the domain organization of SIN3B. Together, these findings shed light on the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.

systems biology↗

bioRxiv2018

Rewiring of a SMAD4 Protein Interaction Network by Breast Tumor Kinase Mediated Phosphorylation

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Interestingly, our search for signaling pathways regulated by BRK, a non-receptor protein tyrosine kinase that is up-regulated in [~]80% of invasive ductal breast tumors, led us to discover that BRK competitively binds and phosphorylates SMAD4, and regulates TGF-{beta}/ SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F), phosphorylates SMAD4 resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. In agreement, we also observed an inverse protein expression pattern of BRK and SMAD4 in a panel of breast cancer cell lines and breast tumors. Activated BRK mediated degradation of SMAD4 causes the repression of tumor suppressor genes FRK that was associated with increased expression of mesenchymal markers and decreased cell adhesion ability. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers. Therefore, our data propose that combination therapies which includes targeting activated BRK signaling may synergize the benefits in the treatment of SMAD4 deficient cancers.

cancer biology↗