Mammalian Homolog Research Articles

Fetal Cartilage Progenitor Cells in the Repair of Osteochondral Defects

Background: Therapies for cartilage restoration are of great interest, but current options provide limited results. In salamanders, interzone (IZN) tissue can regenerate large joint lesions. The mammalian homolog to this tissue exists during fetal development and exhibits remarkable chondrogenesis in vitro. This study analyzed the potential of equine IZN and adjacent anlagen (ANL) cells to regenerate osteochondral defects. Methods: Osteochondral defects were created in the knee of immunosuppressed rats and were grafted with cell pellets from either equine fetal IZN, equine fetal ANL, adult fibroblasts, or adult chondrocytes, or they were left untreated. Osteochondral repair was assessed after 2, 6, and 16 weeks. Results: Untreated lesions unexpectedly failed to represent critical-sized defects and at 2 weeks exhibited new subchondral bone covered by a fibrocartilage layer that thinned over time. Fibroblast-treated defects filled with soft fibrous tissue. Chondrocyte-treated repair tissue exhibited strong proteoglycan and COL2 staining but poor integration to the adjacent bone. Defects treated with IZN, ANL, or chondrocyte pellets developed hyaline cartilage with increasing safranin-O and collagen II staining over time. IZN and ANL repair tissues exhibited some evidence of zonal architecture such as native cartilage and the best bone integration; nonetheless, they developed exuberant growth, often causing patellar instability and osteoarthritis. Conclusions: IZN or ANL cells exhibited some potential to recapitulate developmental features during cartilage repair. However, identifying regulatory determinants of IZN and ANL-derived overgrowths is necessary. Clinical Relevance: Studies grafting IZN or ANL tissues in larger animal models with regular immune functions may provide additional insights into improving osteochondral regeneration.

Selenoprotein O Promotes Melanoma Metastasis and Regulates Mitochondrial Complex II Activity.

Evolutionarily conserved selenoprotein O (SELENOO) catalyzes a post-translational protein modification known as AMPylation that is essential for the oxidative stress response in bacteria and yeast. Given that oxidative stress experienced in the blood limits survival of metastasizing melanoma cells, SELENOO might be able to impact metastatic potential. However, further work is needed to elucidate the substrates and functional relevance of the mammalian homologue of SELENOO. Here, we revealed that SELENOO promotes cancer metastasis and identified substrates of SELENOO in mammalian mitochondria. In patients with melanoma, high SELENOO expression was correlated with metastasis and poor overall survival. In a murine model of spontaneous melanoma metastasis, SELENOO deficiency significantly reduced metastasis to distant visceral organs, which could be rescued by treatment with the antioxidant N-acetylcysteine. Mechanistically, SELENOO AMPylated multiple mitochondrial substrates, including succinate dehydrogenase subunit A, one of the four key subunits of mitochondrial complex II. Consistently, SELENOO-deficient cells featured increased mitochondrial complex II activity. Together, these findings demonstrate that SELENOO deficiency limits melanoma metastasis by modulating mitochondrial function and oxidative stress.

Drosulfakinin signaling encodes early-life memory for adaptive social plasticity.

Drosophila establishes social clusters in groups, yet the underlying principles remain poorly understood. Here we performed a systemic analysis of social network behavior (SNB) that quantifies individual social distance (SD) in a group over time. The SNB assessment in 175 inbred strains from the Drosophila Genetics Reference Panel showed a tight association of short SD with long developmental time, low food intake, and hypoactivity. The developmental inferiority in short-SD individuals was compensated by their group culturing. By contrast, developmental isolation silenced the beneficial effects of social interactions in adults and blunted the plasticity of SNB under physiological challenges. Transcriptome analyses revealed genetic diversity for SD traits, whereas social isolation reprogrammed select genetic pathways, regardless of SD phenotypes. In particular, social deprivation suppressed the expression of the neuropeptide Drosulfakinin (Dsk) in three pairs of adult brain neurons. Male-specific DSK signaling to Cholecystokinin-like receptor 17D1 mediated the SNB plasticity. In fact, transgenic manipulations of the DSK neuron activity were sufficient to imitate the state of social experience. Given the functional conservation of mammalian Dsk homologs, we propose that animals may have evolved a dedicated neural mechanism to encode early-life experience and transform group properties adaptively.

Sdd3 regulates the biofilm formation of Candida albicans via the Rho1-PKC-MAPK pathway.

Candida albicans, the most frequently isolated fungal pathogen in humans, forms biofilms that enhance resistance to antifungal drugs and host immunity, leading to frequent treatment failure. Understanding the molecular mechanisms governing biofilm formation is crucial for developing anti-biofilm therapies. In this study, we conducted a genetic screen to identify novel genes that regulate biofilm formation in C. albicans. One identified gene is ORF19.6693, a homolog of the Saccharomyces cerevisiae SDD3 gene. The sdd3∆/∆ mutant exhibited severe defects in biofilm formation and significantly reduced chitin content in the cell wall. Overexpression of the constitutively active version of the Rho1 GTPase Rho1G18V, an upstream activator of the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) cell-wall integrity pathway, rescued these defects. Affinity purification, mass spectrometry, and co-immunoprecipitation revealed Sdd3's physical interaction with Bem2, the GTPase-activating protein of Rho1. Deletion of SDD3 significantly reduced the amount of the active GTP-bound form of Rho1, thereby diminishing PKC-MAPK signaling and downregulating chitin synthase genes CHS2 and CHS8. Taken together, our studies identify a new biofilm regulator, Sdd3, in C. albicans that modulates Rho1 activity through its inhibitory interaction with Bem2, thereby regulating the PKC-MAPK pathway to control chitin biosynthesis, which is critical for biofilm formation. As an upstream component of the pathway and lacking a homolog in mammals, Sdd3 has the potential to serve as an antifungal target for biofilm infections.IMPORTANCEThe human fungal pathogen Candida albicans is categorized as a critical priority pathogen on the World Health Organization's Fungal Priority Pathogens List. A key virulence attribute of this pathogen is its ability to form biofilms on the surfaces of indwelling medical devices. Fungal cells in biofilms are highly resistant to antifungal drugs and host immunity, leading to treatment failure. This study conducted a genetic screen to discover novel genes that regulate biofilm formation. We found that deletion of the SDD3 gene caused severe biofilm defects. Sdd3 negatively regulates the Rho1 GTPase, an upstream activator of the protein kinase C-mitogen-activated protein kinase pathway, through direct interaction with Bem2, the GTPase-activating protein of Rho1, resulting in a significant decrease in chitin content in the fungal cell wall. This chitin synthesis defect leads to biofilm formation failure. Given its essential role in biofilm formation, Sdd3 could serve as an antifungal target for biofilm infections.

Proteolytic regulation of mitochondrial magnesium channel MRS2 by m-AAA protease and prohibitin complex.

Mitochondrial membrane phospholipid cardiolipin is essential for the stability of several inner mitochondrial membrane protein complexes. We recently showed that the abundance of mitochondrial magnesium channel MRS2 is reduced in models of Barth syndrome, an X-linked genetic disorder caused by a remodeling defect in cardiolipin. However, the mechanism underlying the reduced abundance of MRS2 in cardiolipin-depleted mitochondria remained unknown. In this study, we utilized yeast mutants of mitochondrial proteases to identify an evolutionarily conserved m-AAA protease, Yta10/Yta12, responsible for degrading Mrs2. The activity of m-AAA protease is regulated by the inner mitochondrial membrane scaffolding complex prohibitin, and consistent with this role, we find that Mrs2 turnover is increased in yeast prohibitin mutants. Importantly, we find that deleting Yta10 in cardiolipin-deficient yeast cells restores the steady-state levels of Mrs2 to the wild-type cells, and the knockdown of AFG3L2, a mammalian homolog of Yta12, increases the abundance of MRS2 in a murine muscle cell line. Thus, our work has identified the m-AAA protease/prohibitin complex as an evolutionarily conserved regulator of Mrs2 that can be targeted to restore Mrs2 abundance in cardiolipin-depleted cells.

LC3B: A microtubule-associated protein influences disease progression and prognosis.

Microtubule-associated protein 1 light chain 3B (MAP1LC3B, also known as LC3B) is a mammalian homolog of the autophagy-related protein 8 (ATG8) family. It plays a crucial role in cellular autophagy and is involved in several vital biological processes, including apoptosis and differentiation. Additionally, LC3B regulates immune responses. Due to its close association with malignant tumors and neurodegenerative diseases, and its potential as a prognostic indicator and therapeutic target, LC3B has become a significant research focus. This article aims to provide a comprehensive and systematic understanding of LC3B's role and mechanisms in autophagy, its impact on apoptosis and the underlying mechanisms, its regulation of cellular differentiation and transdifferentiation, its modulation of immune and inflammatory responses, the influence of upstream regulatory factors on LC3B's function, and its relevance to disease diagnosis, treatment, and prognosis. The goal is to establish a solid foundation for understanding LC3B's role in cellular processes and its regulatory mechanisms.

Nuclear receptor E75/NR1D2 promotes tumor malignant transformation by integrating Hippo and Notch pathways

Hormone therapy resistance and the ensuing aggressive tumor progression present a significant clinical challenge. However, the mechanisms underlying the induction of tumor malignancy upon inhibition of steroid hormone signaling remain poorly understood. Here, we demonstrate that Drosophila malignant epithelial tumors show a similar reduction in ecdysone signaling, the main steroid hormone pathway. Our analysis of ecdysone-induced downstream targets reveals that overexpression of the nuclear receptor E75, particularly facilitates the malignant transformation of benign tumors. Genome-wide DNA binding profiles and biochemistry data reveal that E75 not only binds to the transcription factors of both Hippo and Notch pathways, but also exhibits widespread co-binding to their target genes, thus contributing to tumor malignancy. We further validated these findings by demonstrating that depletion of NR1D2, the mammalian homolog of E75, inhibits the activation of Hippo and Notch target genes, impeding glioblastoma progression. Together, our study unveils a novel mechanism by which hormone inhibition promotes tumor malignancy, and describes an evolutionarily conserved role of the oncogene E75/NR1D2 in integration of Hippo and Notch pathway activity during tumor progression.

Biochemical Analysis of the Regulatory Role of Gαo in the Conformational Transitions of Drosophila Pins.

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gαi·GDP, Pins has been found to associate with both Drosophila Gαi (dGαi) and Gαo (dGαo) isoforms. Moreover, dGαo was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of dGαo on the conformational states of Pins remains unclear, despite extensive investigations into the Gαi·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and dGαo in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded dGαo. Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of dGαo·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between dGαi and Pins-GLs, thus implying the pivotal role of dGαo as the principal participant in the ACD pathway associated with Pins.

Structural Perspective of the Double-Stranded RNA Transport Mechanism by SID-1 Family Proteins.

The systemic RNA interference defective 1 (SID-1) family proteins are putative double-stranded RNA (dsRNA) transporters. Two mammalian homologs, SIDT1 and SIDT2 have been linked to many functions such as innate immune responses, microRNA uptake and lysosomal degradation of RNA/DNA whereas Caenorhabditis elegans SID-1 is essential for systemic RNA interference. However, dsRNA uptake mechanism is largely unknown. In this review, we discuss our current understanding of the molecular functions of SID-1 family proteins at a structure level, which highlights recent structural studies.

Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex.

The phosphoribosyl pyrophosphate synthetase (PRPS) enzyme conducts a chokepoint reaction connecting central carbon metabolism and nucleotide production pathways, making it essential for life1,2. Here, we show that the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes, and we trace the evolutionary origins and define the individual functions of each of the five mammalian PRPS homologs - three isozymes (one testis-restricted)3,4 and two non-enzymatic associated proteins (APs)5,6 - which we demonstrate operate together as a large molecular weight complex capable of attaining a heterogeneous array of functional multimeric configurations. Employing a repertoire of isogenic fibroblast clones in all viable individual or combinatorial assembly states, we define preferential interactions between subunits, and we show that cells lacking PRPS2, PRPSAP1, and PRPSAP2 render PRPS1 into aberrant homo-oligomeric assemblies with diminished metabolic flux and impaired proliferative capacity. We demonstrate how numerous evolutionary innovations in the duplicated genes have created specialized roles for individual complex members and identify translational control mechanisms that enable fine-tuned regulation of PRPS assembly and activity, which provide clues into the positive and negative selective pressures that facilitate metabolic flexibility and tissue specialization in advanced lifeforms. Collectively, our study demonstrates how evolution has transformed a single PRPS gene into a multimeric complex endowed with functional and regulatory features that govern cellular biochemistry.

Novel therapies for pediatric low grade glioma.

Current biological findings provide new insights into the genetics driving growth of low-grade gliomas in pediatric patients. This has provided new targets for novel therapies. The purpose of this paper is to review novel therapies for pediatric low-grade gliomas that have been published in the past 24 months. Low-grade gliomas are often driven by mitogen activated protein kinase (MAPK) alterations either with BRAF V600E point mutations or BRAF fusions. Current advances have also highlighted novel fusions of fibroblast growth factor receptor (FGFR), myeloblastosis family of transcription factors (MYB), meningioma 1 tumor suppressor (MN1), neurotrophic receptor kinase family of receptors (NTRK), Kristen RAS (Rat Sarcoma Virus) oncogene homolog in mammals (KRAS), Receptor tyrosine kinase ROS proto oncogene 1 (ROS1), protein kinase C alpha (PRKCA), and platelet derive growth factor receptor (PDGFR) amplification. Novel therapies have been employed and are showing encouraging results in pediatric low-grade gliomas. Current trials are underway with newer generation pan RAF inhibitors and mitogen activated protein kinase - kinase (MEK) inhibitors. Other early phase clinical trials have provided safety data in pediatric patients targeting FGFR fusion, NTRK fusion, PDGFR amplification and ROS1 mutations. Historical treatment options in pediatric low-grade gliomas have utilized surgery, radiation therapy and conventional chemotherapy. Recently greater insight into their biology has found that alterations in MAPK driven pathways are often the hallmark of tumorigenesis. Targeting these novel pathways has led to tumor control and shrinkage without the use of conventional chemotherapy. Caution should be taken however, since these treatment options are still novel, and we do not fully appreciate the long-term effects. Nonetheless a new era of targeted medicine is here.

Structural Analysis of Mammalian Sialic Acid Esterase

Sialic acid esterase (SIAE) catalyzes the removal of O-acetyl groups from sialic acids found on cell surface glycoproteins to regulate cellular processes such as B cell receptor signalling and apoptosis. Loss-of-function mutations in SIAE are associated with several common autoimmune diseases including Crohn’s, ulcerative colitis, and arthritis. To gain a better understanding of the function and regulation of this protein, we determined crystal structures of SIAE from three mammalian homologs, including an acetate bound structure. The structures reveal that the catalytic domain adopts the fold of the SGNH hydrolase superfamily. The active site is composed of a catalytic dyad, as opposed to the previously reported catalytic triad. Attempts to determine a substrate-bound structure yielded only the hydrolyzed product acetate in the active site. Rigid docking of complete substrates followed by molecular dynamics simulations revealed that the active site does not form specific interactions with substrates, rather it appears to be broadly specific to accept sialoglycans with diverse modifications. Based on the acetate bound structure, a catalytic mechanism is proposed. Structural mapping of disease mutations reveals that most are located on the surface of the enzyme and would only cause minor disruptions to the protein fold, suggesting that these mutations likely affect binding to other factors. These results improve our understanding of SIAE biology and may aid in the development of therapies for autoimmune diseases and cancer.

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis.

Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 μM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

Abstract We089: Numb Family Proteins in Epicardium Regulate Epicardial Cells Differentiation and Ventricular Patterning During Development

Background: Numb Family Proteins (NFPs), specifically two mammalian homologs Numb (denoted by Nb ) and Numblike (denoted by Nl ), have been evident to regulate essential cardiac functions during development. Previous studies reported NFPs as integral for epicardial cells (EpiCs) epithelial mesenchymal-transition (EMT). However, whether NFPs in epicardium regulate EpiCs differentiation to affect cardiac morphogenesis have not been explored. In this study, we investigated how NFPs in epicardium regulate EpiCs EMT and differentiation to modulate cardiac development and subsequently affect cardiac maintenance in adult stages. Method: Using mCherry:Numb knockin mouse model we observed enrichment of Numb in epicardium. We deleted Nb and Nl in epicardium via Tbx18 Cre/+ lineage and refer to this as EDKO. We harvested hearts at embryonic day 15.5/18.5 and assessed morphological and mechanistic differences between control and EDKOs. Conventional and Speckle tracking Echocardiography were used to examine adult heart structure and functions. Results: Our findings revealed that EDKO embryonic hearts display specific morphology with extended left ventricles. We found a surprising difference between female and male EDKOs in terms of survival to adulthood. Reduced EpiCs EMT and Cardiac Fibroblasts (CFs) migration distances, changes in CFs distribution have been observed in EDKO developing hearts. Reduced expression of smooth muscle cells (SMCs) and pericytes also was noticed in EDKOs. Strikingly, EDKO embryonic hearts show defects in ventricular compaction, ventricular wall patterning and in cell proliferation. Furthermore, when survived to adult stage, EDKO hearts revealed significant structural and functional changes over their aging. While digging into regulatory pathways, we found changes in Fibroblast growth factor receptors (Fgfrs)/p-Erk pathway and in activation of Notch1 in EDKO hearts. Conclusion: NFPs in epicardium regulate EpiCs differentiation into CFs and SMCs, and regulate ventricular wall morphogenesis during development. NFPs are also required to maintain cardiac structure and function at adult stages. Fgfrs/p-Erk pathway and Notch1 could be the possible mechanism behind cardiac morphogenetic regulation by NFPs in epicardium. Future aims: Further studies will identify downstream targets of NFPs’ regulation of EpiCs differentiation and ventricular wall patterning in embryonic hearts, and mechanistic pathways of epicardial NFPs’ role in adult hearts.

LYSMD proteins promote activation of Rab32-family GTPases for lysosome-related organelle biogenesis.

Lysosome-related organelles (LROs) are specialized lysosomes with cell type-specific roles in organismal homeostasis. Dysregulation of LROs leads to many human disorders, but the mechanisms underlying their biogenesis are not fully understood. Here, we identify a group of LYSMD proteins as evolutionarily conserved regulators of LROs. In Caenorhabditis elegans, mutations of LMD-2, a LysM domain-containing protein, reduce the levels of the Rab32 GTPase ortholog GLO-1 on intestine-specific LROs, the gut granules, leading to their abnormal enlargement and defective biogenesis. LMD-2 interacts with GLO-3, a subunit of GLO-1 guanine nucleotide exchange factor (GEF), thereby promoting GLO-1 activation. Mammalian homologs of LMD-2, LYSMD1, and LYSMD2 can functionally replace LMD-2 in C. elegans. In mammals, LYSMD1/2 physically interact with the HPS1 subunit of BLOC-3, the GEF of Rab32/38, thus promoting Rab32 activation. Inactivation of both LYSMD1 and LYSMD2 reduces Rab32 activation, causing melanosome enlargement and decreased melanin production in mouse melanoma cells. These findings provide important mechanistic insights into LRO biogenesis and functions.

Tissue-specific knockdown of OMM protein via GFP nanobody-mediated degradation

Mitochondria, with their diverse morphologies across tissues, hint at a unique function based on location. For instance, outer mitochondrial membrane (OMM) proteins are critical for various mitochondrial activities, including regulating mitochondrial dynamics, ion homeostasis, and protein translocation. This study introduces a green fluorescent protein (GFP) nanobody-mediated protein degradation (G-DEG) system to investigate tissue-specific mitochondrial functions in Caenorhabditis elegans and potential other model systems. G-DEG combines CRISPR-Cas9 GFP knock-in with ZIF-1-mediated protein degradation, leveraging the high specificity of antigen–antibody recognition for precise manipulation across species. We demonstrate the G-DEG system by targeting FZO-1, a mammalian homolog of MAN1/2, which is essential for mitochondrial fusion. Our protocol includes CRISPR-Cas9-mediated fzo-1:GFP knock-in and the construction of tissue-specific GFP nanobody degradation plasmids for the epidermis, muscle, and neurons. Injection of these plasmids into wild-type C. elegans and subsequent crossbreeding with the fzo-1:GFP knock-in strain allows for effective FZO-1 targeting, providing tissue-specific insights into mitochondrial protein function. Overall, G-DEG emerges as a powerful and versatile tool for tissue-specific knockdown of OMM proteins, paving the way for advanced studies on their diverse biological functions.

Crystal structure of l-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates

l-2-Keto-3-deoxyfuconate 4-dehydrogenase (l-KDFDH) catalyzes the NAD+-dependent oxidization of l-2-keto-3-deoxyfuconate (l-KDF) to l-2,4-diketo-3-deoxyfuconate (l-2,4-DKDF) in the non-phosphorylating l-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of l-KDF. On the other hand, BDH2, a mammalian homolog with l-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-l-proline (C4LHyp) with the cyclic structure. We found that l-KDFDH and BDH2 utilize C4LHyp and l-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of l-KDFDH from Herbaspirillum huttiense in the ligand-free, l-KDF and l-2,4-DKDF, d-KDP (d-2-keto-3-deoxypentonate; additional substrate), or l-2,4-DKDF and NADH bound forms. In complexed structures, l-KDF, l-2,4-DKDF, and d-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, l-KDFDH showed no activity for l-KDF and d-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of l-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

Using Fluorescent GAP Indicators to Monitor ER Ca2+

AbstractThe endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP‐Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+‐binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5‐8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescenceBasic Protocol 2: Monitoring ER Ca2+Basic Protocol 3: Monitoring ER‐ and cytosolic‐Ca2+Support Protocol: Generation of a stable cell line expressing erGAP3