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1. The Cul3 ubiquitin ligase engages Insomniac as an adaptor to impact sleep and synaptic homeostasis

   https://doi.org/10.1371/journal.pgen.1011574  

   Li, Qiuling; Lim, Kayla Y; Altawell, Raad; Verderose, Faith; Li, Xiling; Dong, Wanying; Martinez, Joshua; Dickman, Dion; Stavropoulos, Nicholas (January 2025, PLOS Genetics)

   Ewer, John (Ed.)

   Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis inDrosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated withCul3mutations. 

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2. Molecular landscape of the fungal plasma membrane and implications for antifungal action

   https://doi.org/10.1038/s41467-025-64171-x  

   Jiang, Jennifer; Keniya, Mikhail V; Puri, Anusha; Zhan, Xueying; Cheng, Jeff; Wang, Huan; Lin, Gigi; Lee, Yun-Kyung; Jaber, Nora; Zhao, Caifeng; et al (December 2025, Nature Communications)

   Abstract Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their native structure and spatial distribution remain poorly characterized. Herein, we employ an integrative approach to investigate the organization of plasma membrane protein complexes inCandida glabrata, focusing on two abundant and essential membrane proteins, the β-(1,3)-glucan synthase (GS) and the proton pump Pma1. We show that treatment with caspofungin, an echinocandin antifungal that targets GS, disrupts the native distribution of membrane protein complexes and alters membrane biophysical properties. Perturbation of the sphingolipid biosynthesis further modulates drug susceptibility, revealing that the lipid environment plays an integral role in membrane protein organization and GS-echinocandin interactions. Our work highlights the importance of characterizing membrane proteins in their native context to understand their functions and inform the development of novel antifungal therapies. 

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3. The Arabidopsis KASH protein SINE3 is involved in male and female gametogenesis

   https://doi.org/10.1007/s00497-024-00508-8  

   Moser, Morgan; Groves, Norman R; Meier, Iris (December 2024, Plant Reproduction)

   Key messageThe Arabidopsis KASH protein SINE3 is involved in male and female gametophyte development, likely affecting the first post-meiotic mitosis in both cases, and is required for full seed set. AbstractLinker of nucleoskeleton and cytoskeleton (LINC) complexes are protein complexes spanning the inner and outer membranes of the nuclear envelope (NE) and are key players in nuclear movement and positioning. Through their roles in nuclear movement and cytoskeletal reorganization, plant LINC complexes affect processes as diverse as pollen tube rupture and stomatal development and function. KASH proteins are the outer nuclear membrane component of the LINC complex, with conserved C-termini but divergent N-terminal cytoplasmic domains. Of the known Arabidopsis KASH proteins, SUN-INTERACTING NUCLEAR ENVELOPE PROTEIN 3 (SINE3) has not been functionally characterized. Here, we show that SINE3 is expressed at all stages of male and female gametophyte development. It is located at the NE in male and female gametophytes. Loss of SINE3 results in a female-derived seed set defect, withsine3mutant ovules arresting at stage FG1. Pollen viability is also significantly reduced, with microspores arresting prior to pollen mitosis I. In addition, sine3mutants have a minor male meiosis defect, with some tetrads containing more than four spores. Together, these results demonstrate that the KASH protein SINE3 plays a crucial role in male and female gametophyte development, likely affecting the first post-meiotic nuclear division in both cases. 

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4. DnaK refolds denatured proteins by actively pulling out their misfolded structural elements

   https://doi.org/10.1101/2025.09.22.677870  

   Marszalek, Piotr E (September 2025, bioRxiv)

   a) Abstract DnaK is a prokaryotic Hsp70 chaperone, with numerous functions in helping to fold nascent polypeptides and more generally in proteostasis. It also restores native structures to heat-shocked proteins in an ATP-hydrolysis-dependent manner. The structures of DnaK complexes with nucleotides, co-chaperones andsmallpeptides have already been resolved. However, there are no structures of DnaK complexes with larger, mostly folded substrates, such as firefly luciferase (Fluc, 61 kDa), which impedes the understanding of the mechanism through which DnaK refolds such large proteins. Here, we generated a model of a DnaK-firefly luciferase complex with Alphafold3, and examined its dynamics with all-atom molecular dynamics simulations. In this complex, Fluc is immobilized under the DnaK alpha-helical lid against the NBD, not the SBDβ, contrary to the data reported in the literature for model peptides. The DnaK lid is positioned strategically over Fluc’s helix 405-411, which we recently determined to be the first (and likely the only) helix melted in Fluc at 42 °C. We simulated the interaction between DnaK and the helix in its native and misfolded state and found that during the lid translocation toward the SBDβ, only the melted helix follows the lid and is actively pulled out from Fluc, while the native helix is not dislocated. These observations suggest a new model for the DnaK chaperone mechanism, where the alpha helical lid forms hydrogen bonds to the protein segment to be structurally tested. Lid pulls out only highly deformable misfolded helices, allowing them to refold into their native structures, and does not pull out those that are correctly folded because they are not deformable. Broader Audience Statementc) DnaK is a model chaperone, which can reactivate thermally denatured proteins. Even though a plethora of significant findings about DnaK structure, dynamics and interactions with its co-chaperone have been accumulated over 30 years, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited the ability of the Alphafold3 platform to generate an atomistic model for a complex between DnaK and Firefly luciferase and used molecular dynamics simulations to directly capture how DnaK may assist denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism. 

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5. Emerging structures and dynamic mechanisms of γ-secretase for Alzheimer’s disease

   https://doi.org/10.4103/NRR.NRR-D-23-01781  

   Miao, Yinglong; Wolfe, Michael S (January 2025, Neural Regeneration Research)

   γ-Secretase, called “the proteasome of the membrane,” is a membrane-embedded protease complex that cleaves 150+ peptide substrates with central roles in biology and medicine, including amyloid precursor protein and the Notch family of cell-surface receptors. Mutations in γ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer’s disease. γ-Secretase has thus served as a critical drug target for treating familial Alzheimer’s disease and the more common late-onset Alzheimer’s disease as well. However, critical gaps remain in understanding the mechanisms of processive proteolysis of substrates, the effects of familial Alzheimer’s disease mutations, and allosteric modulation of substrate cleavage by γ-secretase. In this review, we focus on recent studies of structural dynamic mechanisms of γ-secretase. Different mechanisms, including the “Fit-Stay-Trim,” “Sliding-Unwinding,” and “Tilting-Unwinding,” have been proposed for substrate proteolysis of amyloid precursor protein by γ-secretase based on all-atom molecular dynamics simulations. While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-bound γ-secretase, molecular dynamics simulations on a resolved model of Notch1-bound γ-secretase that was reconstructed using the amyloid precursor protein-bound γ-secretase as a template successfully captured γ-secretase activation for proper cleavages of both wildtype and mutant Notch, being consistent with biochemical experimental findings. The approach could be potentially applied to decipher the processing mechanisms of various substrates by γ-secretase. In addition, controversy over the effects of familial Alzheimer’s disease mutations, particularly the issue of whether they stabilize or destabilize γ-secretase-substrate complexes, is discussed. Finally, an outlook is provided for future studies of γ-secretase, including pathways of substrate binding and product release, effects of modulators on familial Alzheimer’s disease mutations of the γ-secretase-substrate complexes. Comprehensive understanding of the functional mechanisms of γ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer’s disease and perhaps Alzheimer’s disease in general. 

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