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1. Molecular docking to homology models of human and Trypanosoma brucei ERK8 that identified ortholog-specific inhibitors

   https://doi.org/10.1371/journal.pntd.0013487  

   Merski, Matthew; Mackey, Zachary B (September 2025, PLOS Neglected Tropical Diseases)

   Amato, Valdir Sabbaga (Ed.)

   Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a lethal disease caused by two vector-borne parasites:Trypanosoma brucei gambienseandTrypanosoma brucei rhodesiense. The limited number of antitrypanosomal therapies for treating these deadly parasites suffer from toxicity, poor efficacy, and unspecified targets; thus, more and better medicines are needed. We usedin silicomethods to predict features of the bioactive compound AZ960 that make it an ortholog-specific inhibitor for the extracellular-signal regulated kinase 8 ofT. brucei(TbERK8). Our homology models showed that the TbERK8 ATP binding pocket was smaller and more hydrophobic than that of human ERK8 (HsERK8). Molecular docking studies predicted six FDA-approved compounds that would be orthologue-specific inhibitors of HsERK8 or TbERK8. Experimental testing of these compounds identified prednisolone as an HsERK8-specific inhibitor. Sildenafil inhibited TbERK8, as predicted by our binding model. Its impact on TbERK8 activity supports our hypothesis that designing compounds that can exploit differences in the orthologs as buildable scaffolds and expand the repertoire of ortholog-specific antitrypanosomal agents. 

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2. Genomic Occupancy of the Bromodomain Protein Bdf3 Is Dynamic during Differentiation of African Trypanosomes from Bloodstream to Procyclic Forms

   https://doi.org/10.1128/msphere.00023-22  

   Ashby, Ethan; Paddock, Lucinda; Betts, Hannah L.; Liao, Jingwen; Miller, Geneva; Porter, Anya; Rollosson, Lindsey M.; Saada, Carrie; Tang, Eric; Wade, Serenity J.; et al (June 2022, mSphere)

   Phillips, Margaret (Ed.)

   ABSTRACT Trypanosoma brucei , the causative agent of human and animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism during adaptation to each host environment. These changes are reflected in the different transcriptomes of parasites living in each host. However, it remains unclear whether chromatin-interacting proteins help mediate these changes. Bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stages remains unknown. To address this question, we performed cleavage under target and release using nuclease (CUT&RUN) against bromodomain protein 3 (Bdf3) in parasites differentiating from bloodstream to insect forms. We found that Bdf3 occupancy at most loci increased at 3 h following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes with altered transcript levels during differentiation, such as procyclins, procyclin-associated genes, and invariant surface glycoproteins. Most Bdf3-occupied sites are observed throughout differentiation. However, one site appears de novo during differentiation and lies proximal to the procyclin gene locus housing genes essential for remodeling surface proteins following transition to the insect stage. These studies indicate that occupancy of chromatin-interacting proteins is dynamic during life cycle stage transitions and provide the groundwork for future studies on the effects of changes in bromodomain protein occupancy. Additionally, the adaptation of CUT&RUN for Trypanosoma brucei provides other researchers with an alternative to chromatin immunoprecipitation (ChIP). IMPORTANCE The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Existing drugs have significant side effects. Thus, these parasites impose a significant human and economic burden in sub-Saharan Africa, where trypanosomiasis is endemic. T. brucei cycles between the mammalian host and a tsetse fly vector, and parasites undergo huge changes in morphology and metabolism to adapt to different hosts. Here, we show that DNA-interacting bromodomain protein 3 (Bdf3) shows changes in occupancy at its binding sites as parasites transition from the bloodstream to the insect stage. Additionally, a new binding site appears near the locus responsible for remodeling of parasite surface proteins during transition to the insect stage. Understanding the mechanisms behind host adaptation is important for understanding the life cycle of the parasite. 

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3. The conformation of the nSrc specificity-determining loop in the Src SH3 domain is modulated by a WX conserved sequence motif found in SH3 domains

   https://doi.org/10.3389/fmolb.2024.1487276  

   Longshore-Neate, Frederick; Ceravolo, Caroline; Masuga, Cole; Tahti, Elise F; Blount, Jadon M; Smith, Sarah N; Amacher, Jeanine F (December 2024, Frontiers in Molecular Biosciences)

   Cellular signaling networks are modulated by multiple protein-protein interaction domains that coordinate extracellular inputs and processes to regulate cellular processes. Several of these domains recognize short linear motifs, or SLiMs, which are often highly conserved and are closely regulated. One such domain, the Src homology 3 (SH3) domain, typically recognizes proline-rich SLiMs and is one of the most abundant SLiM-binding domains in the human proteome. These domains are often described as quiteversatile, and indeed, SH3 domains can bind ligands in opposite orientations dependent on target sequence. Furthermore, recent work has identified diverse modes of binding for SH3 domains and a wide variety of sequence motifs that are recognized by various domains. Specificity is often attributed to the RT and nSrc loops near the peptide-binding cleft in this domain family, particularly for Class I binding, which is defined as RT and nSrc loop interactions with the N-terminus of the ligand. Here, we used the Src and Abl SH3 domains as a model to further investigate the role of the RT and nSrc loops in SH3 specificity. We created chimeric domains with both the RT and nSrc loop sequences swapped between these SH3 domains, and used fluorescence anisotropy assays to test how relative binding affinities were affected for Src SH3- and Abl SH3-specific ligands. We also used Alphafold–Multimer to model our SH3:peptide complexes in combination with molecular dynamics simulations. We identified a position that contributes to the nSrc loop conformation in Src SH3, the amino acid immediately following a highly conserved Trp that creates a hydrophobic pocket critical for SH3 ligand recognition. We defined this as the WX motif, where X = Trp for Src and Cys for Abl. A broad importance of this position for modulating nSrc loop conformation in SH3 domains is suggested by analyses of previously deposited SH3 structures, multiple sequence alignment of SH3 domains in the human proteome, and our biochemical and computational data of mutant Src and Abl SH3 domains. Overall, our work uses experimental approaches and structural modeling to better understand specificity determinants in SH3 domains. 

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4. PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication

   https://doi.org/10.1073/pnas.1814521115  

   Lin, Yo-Chuen; Wang, Yating; Hsu, Rosaline; Giri, Sumanprava; Wopat, Susan; Arif, Mariam K.; Chakraborty, Arindam; Prasanth, Kannanganattu V.; Prasanth, Supriya G. (December 2018, Proceedings of the National Academy of Sciences)

   RING finger and WD repeat domain-containing protein 3 (RFWD3) is an E3 ligase known to facilitate homologous recombination by removing replication protein A (RPA) and RAD51 from DNA damage sites. Further, RPA-mediated recruitment of RFWD3 to stalled replication forks is essential for interstrand cross-link repair. Here, we report that in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif. PCNA association is critical for the stability of RFWD3 and for DNA replication. Cells lacking RFWD3 show slower fork progression, a prolonged S phase, and an increase in the loading of several replication-fork components on the chromatin. These findings all point to increased frequency of stalled forks in the absence of RFWD3. The S-phase defect is rescued by WT RFWD3, but not by the PIP mutant, suggesting that the interaction of RFWD3 with PCNA is critical for DNA replication. Finally, we observe reduced ubiquitination of RPA in cells lacking RFWD3. We conclude that the stabilization of RFWD3 by PCNA at the replication fork enables the polyubiquitination of RPA and its subsequent degradation for proper DNA replication. 

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5. MotifAnalyzer‐PDZ : A computational program to investigate the evolution of PDZ‐binding target specificity

   https://doi.org/10.1002/pro.3741  

   Valgardson, Jordan; Cosbey, Robin; Houser, Paul; Rupp, Milo; Van Bronkhorst, Raiden; Lee, Michael; Jagodzinski, Filip; Amacher, Jeanine F. (November 2019, Protein Science)

   Abstract Recognition of short linear motifs (SLiMs) or peptides by proteins is an important component of many cellular processes. However, due to limited and degenerate binding motifs, prediction of cellular targets is challenging. In addition, many of these interactions are transient and of relatively low affinity. Here, we focus on one of the largest families of SLiM‐binding domains in the human proteome, the PDZ domain. These domains bind the extreme C‐terminus of target proteins, and are involved in many signaling and trafficking pathways. To predict endogenous targets of PDZ domains, we developedMotifAnalyzer‐PDZ, a program that filters and compares all motif‐satisfying sequences in any publicly available proteome. This approach enables us to determine possible PDZ binding targets in humans and other organisms. Using this program, we predicted and biochemically tested novel human PDZ targets by looking for strong sequence conservation in evolution. We also identified three C‐terminal sequences in choanoflagellates that bind a choanoflagellate PDZ domain, theMonsiga brevicollisSHANK1 PDZ domain (mbSHANK1), with endogenously‐relevant affinities, despite a lack of conservation with the targets of a homologous human PDZ domain, SHANK1. All three are predicted to be signaling proteins, with strong sequence homology to cytosolic and receptor tyrosine kinases. Finally, we analyzed and compared the positional amino acid enrichments in PDZ motif‐satisfying sequences from over a dozen organisms. Overall,MotifAnalyzer‐PDZis a versatile program to investigate potential PDZ interactions. This proof‐of‐concept work is poised to enable similar types of analyses for other SLiM‐binding domains (e.g.,MotifAnalyzer‐Kinase).MotifAnalyzer‐PDZis available athttp://motifAnalyzerPDZ.cs.wwu.edu. 

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