Abstract 2257 Exploring transcription factor regulation by Cdc14 phosphatase in response to cell wall stress in the opportunistic pathogen Candida albicans

Journal of Biological Chemistry · 2025-05-01

Human fungal pathogens result in over 6 million cases of invasive infections and more than 3 million deaths each year.Despite this, research to better understand these pathogens and develop new antifungal drugs lags behind that of other infectious disease classes.Candida albicans is designated a critical priority pathogen by the World Health Organization, as it accounts for the majority of all fungal infections and is associated with a high mortality rate for bloodstream infections.We recently identified a novel cell wall integrity (CWI) role for the protein phosphatase Cdc14 in Candida albicans.When CDC14 is deleted, C. albicans is more susceptible to treatment with echinocandin antifungal drugs that inhibit cell wall synthesis, and virulence is also greatly reduced.However, the role that Cdc14 plays in the CWI pathway, including its substrates, are not well understood.In cells lacking Cdc14, the CWI MAP kinase pathway is still activated normally in response to cell wall stress, but expression of downstream CWI target genes, such as ECM331 and PGA31, fails to induce.This implies that Cdc14 functions downstream of the MAP kinase activation step in the CWI signaling pathway, but upstream of target gene transcription.We hypothesize that Cdc14 is activated by cell wall stress to dephosphorylate the transcription factors (TFs) responsible for the CWI response, leading to their nuclear import.In the model fungus S. cerevisiae, Cdc14 de-phosphorylates several TFs during the cell cycle to activate their nuclear import, including Swi6, which also plays a role in the CWI response.Using a bioinformatic search algorithm I identified a small set of TFs with candidate Cdc14-regulated nuclear localization signals, and I predict one or more will localize to the nucleus in response to cell wall stress in a Cdc14-dependent manner.To test this, I designed strains expressing each candidate substrate tagged with GFP from its natural promoter, as well as phosphosite mutants predicted to bypass the requirement for Cdc14 de-phosphorylation.I am comparing localization of the wild-type and mutant proteins with and without 1) Cdc14 and 2) echinocandin drug treatment.I expect this work will provide mechanistic insight into how Cdc14 controls the response to cell wall stress in Candida albicans.

Abstract 2208 Characterization of an expanded Cdc14 phosphatase family in the ciliated protozoan Tetrahymena thermophila

Journal of Biological Chemistry · 2025-05-01

Cdc14 is a highly conserved protein phosphatase across eukaryotes, including humans, but notably absent in angiosperms. Initially identified in baker's yeast as a key regulator of cell division, Cdc14 plays different roles in humans. Mutations in human Cdc14A have been linked to male infertility and hearing loss due to defects in cilia maintenance and function. However, the mechanisms underlying hCdc14A's role in ciliary structure and function and why hCdc14A mutations lead to these defects remain unclear.

Abstract 1679 Characterization of the role of Cdc14 in hyphal development in Candida albicans

Journal of Biological Chemistry · 2025-05-01

Cdc14 phosphatase is a highly conserved member of the dual-specificity subfamily of the protein tyrosine phosphatases. In Candida albicans, an opportunistic human pathogen, hyphal development is required for virulence and previous work in our lab has shown that CDC14 mutant strains form defective hyphae. As a result, these CDC14 mutants are avirulent and Cdc14 may be a useful antifungal drug target. The long-term goal of our lab is to characterize the molecular mechanisms by which Cdc14 controls hyphal development in C.

Abstract 1677 Investigating the role of HGC1 in hyphal development and maintenance using auxin-inducible degrataion in Candida albicans

Journal of Biological Chemistry · 2025-05-01

Candida albicans is a fungal species found in the gastrointestinal tract and mucosal membranes.It is usually harmless but can become an opportunistic pathogen in people who are immune-compromised.In rare cases, it can cause invasive candidiasis, when the fungus spreads to internal organs such as the liver and kidneys, often disseminating through the bloodstream.The mortality rate of invasive candidiasis is 10-50% depending on many internal and external factors.C. albicans has two morphological states: the yeast state and the filamentous state.Both are required for pathogenesis.Hgc1 is an essential protein for promoting the hyphal state.Hgc1 is only expressed in hyphae and forms a complex with cyclin-dependent kinase (Cdk1) to phosphorylate target proteins that promote hyperpolarized growth and suppress cell separation.However only a few protein substrates of Hgc1-Cdk have been identified.Since hyphal growth in C. albicans is required for virulence, substrates regulated by Hgc1 might be useful targets for antifungal drugs.The objective of this project is to identify Hgc1-Cdk substrates to better understand how it promotes and maintains the hyphal state.To characterize Hgc1 function in hyphal maintenance, we need a tool to disrupt it after hyphae have started to develop, and we chose the auxin-inducible degradation (AID) system for this.The AID system is used to rapidly degrade a target protein of interest to observe the resulting phenotypic effects.Target degradation is activated by adding the plant hormone auxin to the culture media.My immediate objective has been to test if auxin-induced Hgc1 degradation is sufficient to phenocopy the hyphal defects of hgc1 deletion strains and if hyphal growth is rapidly arrested upon auxin addition.If so, then AID can be used to study the mechanisms by which Hgc1 maintains polarized hyphal growth and prevents cell separation.I am testing suitability of the AID system by inducing hyphal growth and then adding auxin to our HGC1-AID strain and observing the effects by light microscopy at different time points.I am comparing the length and number of hyphal cells over time with and without auxin.The expected result is that auxin treatment will cause hyphal elongation to stop, and cell separation to begin.

Structure of E6AP in complex with HPV16-E6 and p53 reveals a novel ordered domain important for E3 ligase activation

Structure · 2025-01-15 · 4 citations

Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition

Structural Dynamics · 2025-09-01

Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo- electron microscopy (cryo-EM) structures of the TnsC-TnsD- att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7- like transposons, with implications for improving the precision and efficiency of their genome-editing applications.

Abstract 2248 Characterizing Cdc14 Phosphatase Function in Cell Wall Stress Response in Candida albicans

Journal of Biological Chemistry · 2025-05-01

Candida albicans is a human fungal pathogen responsible for 45–50% of fungal infections, with rising resistance to conventional antifungal therapies. This growing resistance, coupled with high mortality rates, underscores the need for therapeutic strategies targeting novel fungal virulence mechanisms. The fungal cell wall is an essential structure, vital for maintaining cellular integrity, mediating interactions with the host immune system, providing a barrier against environmental stress, and is the target of current anti-fungal therapies.

Breaking the Boundaries Collective – A Manifesto for Relationship-based Practice

Ethics and Social Welfare · 2024-01-02 · 5 citations

This paper argues that professionals who make boundary-related decisions should be guided by relationship-based practice. In our roles as service users and professionals, drawing from our lived experiences of professional relationships, we argue we need to move away from distance-based practice. This includes understanding the boundary stories and narratives that exist for all of us -including the people we support, other professionals, as well as the organisations and systems within which we work. When we are dealing with professional boundary issues, we should centre relationship-building skills that are central to many other aspects of our work. Skills that foster relationships at all levels -between professionals, service users, and services -need to be revalued. Our final recommendation is to create, develop, and foster safer spaces within and outside of organisations, as well as inter-professionally, for the discussion and exploration of boundary-related issues and practice. We are interested in hearing from those with experiences of being marginalised by boundaries so that they can inform a reshaping of our collective ideas around boundary related practices. To foster relationshipbased practices in organisations, we have outlined several recommendations here; however, we recognize that these do not go far enough, and that collective action is needed to inform systemic change.

Structure of E6AP in Complex with HPV16-E6 and p53 Reveals a Novel Ordered Domain Important for E3 Ligase Activation

SSRN Electronic Journal · 2024-01-01

Cdc14 phosphatases use an intramolecular pseudosubstrate motif to stimulate and regulate catalysis

Journal of Biological Chemistry · 2024-08-08 · 1 citations

Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTPs) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14, we discovered a short sequence in the disordered C terminus, distal to the catalytic domain, which mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.