Protein Family Research Articles

The merger of two major families of proteins that regulate cellular processes

In this issue of Structure, Oot and Wilkens1 present new mechanistic insights to finally merge the function of V-ATPase and TLDc domain proteins. They show that TLDc proteins directly affect V-ATPase activity and assembly, expanding our understanding of how V-ATPase and TLDc proteins exert a plethora of biological functions.

Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

YTH domain family protein 3 accelerates non-small cell lung cancer immune evasion through targeting CD8+ T lymphocytes

Immune evasion is one of the critical hallmarks of malignant tumors, especially non-small cell lung cancer (NSCLC). Emerging findings have illustrated the roles of N6-methyladenosine (m6A) on NSCLC immune evasion. Here, this study investigated the function and underlying mechanism of m6A reader YTH domain family protein 3 (YTHDF3) on NSCLC immune evasion. YTHDF3 was found to be highly expressed in NSCLC tissue and act as an independent prognostic factor for overall survival. Functionally, up-regulation of YTHDF3 impaired the CD8+ T antitumor activity to deteriorate NSCLC immune evasion, while YTHDF3 silencing recovered the CD8+ T antitumor activity to inhibit immune evasion. Besides, YTHDF3 up-regulation reduced the apoptosis of NSCLC cells. Mechanistically, PD-L1 acted as the downstream target for YTHDF3, and YTHDF3 could upregulate the transcription stability of PD-L1 mRNA. Overall, YTHDF3 targeted PD-L1 to promote NSCLC immune evasion partially through escaping effector cell cytotoxicity CD8+ T mediated killing and antitumor immunity. In summary, this study provides an essential insight for m6A modification on CD8+ T cell-mediated antitumor immunity in NSCLC, which might inspire an innovation for lung cancer tumor immunotherapy.

Structural Analysis of the ESCRT-III Regulator Lethal(2) Giant Discs/Coiled-Coil and C2 Domain-Containing Protein 1 (Lgd/CC2D1)

Members of the LGD/CC2D1 protein family contain repeats of the family-defining DM14 domains. Via this domain, they interact with members of the CHMP family, which are essential for the ESCRT machinery-mediated formation of intraluminal vesicles during endosome maturation. Here, we investigate the requirement of the DM14 domains for the function of Lgd in detail. We found that although both odd-numbered DM14s can act in a functionally redundant manner, the redundancy is not complete and both contribute to the full function of Lgd. Our analysis indicates that some of the AAs that form the KARRxxR motif of the onDM14s are not exchangeable by similarly charged AAs without loss of function, indicating that they not only provide charge, but also fulfil structural roles. Furthermore, we show that the region of Lgd between DM14-4 and the C2 domain as well as its C-terminal region to the C2 domain are important for protein stability/function. Moreover, we analysed the importance of AAs that are conserved in all DM14 domains. Finally, our analysis of the C. elegans ortholog of Lgd revealed that it has only one DM14 domain that is functionally equivalent to the onDM14s. Altogether, the results further the understanding of how Lgd family members regulate the ESCRT machinery.

Fine-tuning Protein Embeddings for Functional Similarity Evaluation.

Protein's with unknown function are frequently compared to better characterized relatives, either using sequence similarity, or recently through similarity in a learned embedding space. Through comparison, protein sequence embeddings allow for interpretable and accurate annotation of proteins, as well as for downstream tasks such as clustering for unsupervised discovery of protein families. However, it's unclear whether embeddings can be deliberately designed to improve their use in these downstream tasks. We find that for functional annotation of proteins, as represented by Gene Ontology (GO) terms, direct fine-tuning of language models on a simple classification loss has an immediate positive impact on protein embedding quality. Fine-tuned embeddings show stronger performance as representations for K-nearest neighbor classifiers, reaching stronger performance for GO annotation than even directly comparable fine-tuned classifiers, while maintaining interpretability through protein similarity comparisons. They also maintain their quality in related tasks, such as rediscovering protein families with clustering. github.com/mofradlab/go_metric. Supplementary data are available at Bioinformatics online.

Fine-tuning Protein Embeddings for Functional Similarity Evaluation.

Protein's with unknown function are frequently compared to better characterized relatives, either using sequence similarity, or recently through similarity in a learned embedding space. Through comparison, protein sequence embeddings allow for interpretable and accurate annotation of proteins, as well as for downstream tasks such as clustering for unsupervised discovery of protein families. However, it's unclear whether embeddings can be deliberately designed to improve their use in these downstream tasks. We find that for functional annotation of proteins, as represented by Gene Ontology (GO) terms, direct fine-tuning of language models on a simple classification loss has an immediate positive impact on protein embedding quality. Fine-tuned embeddings show stronger performance as representations for K-nearest neighbor classifiers, reaching stronger performance for GO annotation than even directly comparable fine-tuned classifiers, while maintaining interpretability through protein similarity comparisons. They also maintain their quality in related tasks, such as rediscovering protein families with clustering. github.com/mofradlab/go_metric. Supplementary data are available at Bioinformatics online.

Explaining Conformational Diversity in Protein Families through Molecular Motions

Proteins play a central role in biological processes, and understanding their conformational variability is crucial for unraveling their functional mechanisms. Recent advancements in high-throughput technologies have enhanced our knowledge of protein structures, yet predicting their multiple conformational states and motions remains challenging. This study introduces Dimensionality Analysis for protein Conformational Exploration (DANCE) for a systematic and comprehensive description of protein families conformational variability. DANCE accommodates both experimental and predicted structures. It is suitable for analysing anything from single proteins to superfamilies. Employing it, we clustered all experimentally resolved protein structures available in the Protein Data Bank into conformational collections and characterized them as sets of linear motions. The resource facilitates access and exploitation of the multiple states adopted by a protein and its homologs. Beyond descriptive analysis, we assessed classical dimensionality reduction techniques for sampling unseen states on a representative benchmark. This work improves our understanding of how proteins deform to perform their functions and opens ways to a standardised evaluation of methods designed to sample and generate protein conformations.

Structure of the flotillin complex in a native membrane environment

In this study, we used cryoelectron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm at its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C terminus, which forms two helical layers linked by a β-strand, and coiled-coil domains that enable strong charge-charge intersubunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillin's roles in cell biology.

Structure of the flotillin complex in a native membrane environment

In this study, we used cryoelectron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm at its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C terminus, which forms two helical layers linked by a β-strand, and coiled-coil domains that enable strong charge-charge intersubunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillin's roles in cell biology.

N - acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in M. marinum.

Acetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

Genome-Wide Identification and Bioinformatics Analysis of the FK506 Binding Protein Family in Rice

Genome-Wide Identification and Bioinformatics Analysis of the FK506 Binding Protein Family in Rice

The C-terminal sequences of Bcl-2 family proteins mediate interactions that regulate cell death.

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP.

Insight into thecardioprotective effects of melatonin: shining a spotlight on intercellular Sirt signaling communication.

Cardiovascular diseases (CVDs) are the leading causes of death and illness worldwide. While there have been advancements in the treatment of CVDs using medication and medical procedures, these conventional methods have limited effectiveness in halting the progression of heart diseases to complete heart failure. However, in recent years, the hormone melatonin has shown promise as a protective agent for the heart. Melatonin, which is secreted by the pineal gland and regulates our sleep-wake cycle, plays a role in various biological processes including oxidative stress, mitochondrial function, and cell death. The Sirtuin (Sirt) family of proteins has gained attention for their involvement in many cellular functions related to heart health. It has been well established that melatonin activates the Sirt signaling pathways, leading to several beneficial effects on the heart. These include preserving mitochondrial function, reducing oxidative stress, decreasing inflammation, preventing cell death, and regulating autophagy in cardiac cells. Therefore, melatonin could play crucial roles in ameliorating various cardiovascular pathologies, such as sepsis, drug toxicity-induced myocardial injury, myocardial ischemia-reperfusion injury, hypertension, heart failure, and diabetic cardiomyopathy. These effects may be partly attributed to the modulation of different Sirt family members by melatonin. This review summarizes the existing body of literature highlighting the cardioprotective effects of melatonin, specifically the ones including modulation of Sirt signaling pathways. Also, we discuss the potential use of melatonin-Sirt interactions as a forthcoming therapeutic target for managing and preventing CVDs.

AZGs: a new family of cytokinin transporters.

Cytokinins (CKs) are phytohormones structurally similar to purines that play important roles in various aspects of plant physiology and development. The local and long-distance distribution of CKs is very important to control their action throughout the plant body. Over the past decade, several novel CK transporters have been described, many of which have been linked to a physiological function rather than simply their ability to transport the hormone in vitro. Purine permeases, equilibrative nucleotide transporters and ATP-binding cassette transporters are involved in the local and long-range distribution of CK. In addition, members of the Arabidopsis AZA-GUANINE RESISTANT (AZG) protein family, AZG1 and AZG2, have recently been shown to mediate CK uptake at the plasma membrane and endoplasmic reticulum. Despite sharing ∼50% homology, AZG1 and AZG2 have unique transport mechanisms, tissue-specific expression patterns, and subcellular localizations that underlie their distinct physiological functions. AZG2 is expressed in a small group of cells in the overlying tissue around the lateral root primordia, where its expression is induced by auxins and it is involved in the regulation of lateral root growth. AZG1 is ubiquitously expressed, with high levels in the division zone of the root apical meristem. Here, it binds and stabilises the auxin efflux carrier PIN1, thereby shaping root architecture, particularly under salt stress. This review highlights the latest findings on the protein properties, transport mechanisms and cellular functions of this new family of CK transporters and discusses perspectives for future research in this field.

Identification of Alternatively Spliced Novel Isoforms of Human HSPB8 Gene.

HSPB8 is a heat shock protein belonging to a family of ATP-independent stress proteins called HSPB which are present far and wide in the cells of various organisms. They are committed to protein quality control (PQC) and strive to avert protein aggregation and to procreate a pool of non-native proteins that can be swiftly folded. Their fundamental expression or stress inducibility is regulated by various cis-elements localized in the HSPB regulatory regions. In the current study we have predicted and confirmed two alternatively spliced novel transcripts of HSPB8 gene in liver, brain, and heart. These spliced variants have smaller sizes owing to smaller N terminal regions and showed remarkable changes in their cellular localization. Novel isoform (HSPB8-N1) was predicted to be majorly localized to nuclear region while the reported isoform (HSPB8) and one of the novel isoforms (HSPB8-N2) were predicted to be cytoplasmic in nature. There were many changes observed in the phosphorylation sites of the novel isoforms as well. The newly reported isoforms lack several structural motifs that are essential for various functional endeavors of the HSPB8 protein. In silico analysis of the conceptually translated protein was carried out using various bioinformatics tools to gain an understanding of their properties in order to explore their possible potential in therapeutics.

Genetically-clustered antifungal phytocytokines and receptor protein family members cooperate to trigger plant immune signaling.

Phytocytokines regulate plant immunity by cooperating with cell-surface proteins. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP-ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and molecular physiology assays to functionally characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Despite a low sequence identity, Salix purpurea RISP1 (SpRISP1) shows properties and activities similar to PtRISP1. Both PtRISP1 and SpRISP1 induced a reactive oxygen species (ROS) burst and mitogen-activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 also triggers a rapid stomatal closure in poplar. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with genes coding LRR-RPs required to initiate immune signaling.

Shifts in keratin isoform expression activate motility signals during wound healing.

Keratin intermediate filaments confer structural stability to epithelial tissues, but the reason this simple mechanical function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. If and how this change modulates cellular functions that support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising mechanical stability by activating myosin motors to increase contractile force generation. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state.

Genetically-clustered antifungal phytocytokines and receptor protein family members cooperate to trigger plant immune signaling.

Phytocytokines regulate plant immunity by cooperating with cell-surface proteins. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP-ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and molecular physiology assays to functionally characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Despite a low sequence identity, Salix purpurea RISP1 (SpRISP1) shows properties and activities similar to PtRISP1. Both PtRISP1 and SpRISP1 induced a reactive oxygen species (ROS) burst and mitogen-activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 also triggers a rapid stomatal closure in poplar. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with genes coding LRR-RPs required to initiate immune signaling.

A Review of the Bromodomain and Extraterminal Domain Epigenetic Reader Proteins: Function on Virus Infection and Cancer

The BET (bromodomain and extraterminal domain) family of proteins, particularly BRD4 (bromodomain-containing protein 4), plays a crucial role in transcription regulation and epigenetic mechanisms, impacting key cellular processes such as proliferation, differentiation, and the DNA damage response. BRD4, the most studied member of this family, binds to acetylated lysines on both histones and non-histone proteins, thereby regulating gene expression and influencing diverse cellular functions such as the cell cycle, tumorigenesis, and immune responses to viral infections. Given BRD4’s involvement in these fundamental processes, it is implicated in various diseases, including cancer and inflammation, making it a promising target for therapeutic development. This review comprehensively explores the roles of the BET family in gene transcription, DNA damage response, and viral infection, discussing the potential of targeted small-molecule compounds and highlighting BET proteins as promising candidates for anticancer therapy.

A Review of the Bromodomain and Extraterminal Domain Epigenetic Reader Proteins: Function on Virus Infection and Cancer

The BET (bromodomain and extraterminal domain) family of proteins, particularly BRD4 (bromodomain-containing protein 4), plays a crucial role in transcription regulation and epigenetic mechanisms, impacting key cellular processes such as proliferation, differentiation, and the DNA damage response. BRD4, the most studied member of this family, binds to acetylated lysines on both histones and non-histone proteins, thereby regulating gene expression and influencing diverse cellular functions such as the cell cycle, tumorigenesis, and immune responses to viral infections. Given BRD4’s involvement in these fundamental processes, it is implicated in various diseases, including cancer and inflammation, making it a promising target for therapeutic development. This review comprehensively explores the roles of the BET family in gene transcription, DNA damage response, and viral infection, discussing the potential of targeted small-molecule compounds and highlighting BET proteins as promising candidates for anticancer therapy.