bims-bicyki 2023-08-20 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2023–08–20
fourteen papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne

Individualized nutritional prescription is necessary for patients with autosomal dominant polycystic kidney disease.
Primary cilia in skeletal development and disease.
Prostaglandin E2, osmoregulation and disease progression in autosomal dominant polycystic kidney disease.
Prenatal diagnosis of polycystic kidney caused by biallelic hypomorphic variants in the PKD1 gene.
Cleavage fragments of the C-terminal tail of polycystin-1 are regulated by oxidative stress and induce mitochondrial dysfunction.
Pkhd1cyli/cyli mice have altered renal Pkhd1 mRNA processing and hormonally sensitive liver disease.
RABL2 promotes the outward transition zone passage of signaling proteins in cilia via ARL3.
Ciliary ARL13B prevents obesity in mice.
The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries.
EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO.
Diabetes impairs fracture healing through Foxo1 mediated disruption of ciliogenesis.
Hedgehog target genes regulate lipid metabolism to drive basal cell carcinoma and medulloblastoma.
Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.
The putative protein kinase Stk36 is essential for ciliogenesis and CSF flow by associating with Ulk4.

J Ren Nutr. 2023 Aug 10. pii: S1051-2276(23)00125-5. [Epub ahead of print]

Individualized nutritional prescription is necessary for patients with autosomal dominant polycystic kidney disease.

Yun Kyu Oh.

DOI: https://doi.org/10.1053/j.jrn.2023.08.004
Exp Cell Res. 2023 Aug 11. pii: S0014-4827(23)00299-9. [Epub ahead of print] 113751

Primary cilia in skeletal development and disease.

Neha Quadri, Priyanka Upadhyai.

  Primary cilia are non-motile, microtubule-based sensory organelle present in most vertebrate cells with a fundamental role in the modulation of organismal development, morphogenesis, and repair. Here we focus on the role of primary cilia in embryonic and postnatal skeletal development. We examine evidence supporting its involvement in physiochemical and developmental signaling that regulates proliferation, patterning, differentiation and homeostasis of osteoblasts, chondrocytes, and their progenitor cells in the skeleton. We discuss how signaling effectors in mechanotransduction and bone development, such as Hedgehog, Wnt, Fibroblast growth factor and second messenger pathways operate at least in part at the primary cilium. The relevance of primary cilia in bone formation and maintenance is underscored by a growing list of rare genetic skeletal ciliopathies. We collate these findings and summarize the current understanding of molecular factors and mechanisms governing primary ciliogenesis and ciliary function in skeletal development and disease.

Keywords:  Chondrogenic differentiation; Ciliary signaling; Endochondral ossification; Intraflagellar transport; Mechanotransduction; Osteogenic differentiation; Primary cilia; Primary cilia regulation; Skeletal ciliopathies; Skeletal development; Skeletal dysplasias

DOI:  https://doi.org/10.1016/j.yexcr.2023.113751
Clin J Am Soc Nephrol. 2023 Aug 14.

Prostaglandin E2, osmoregulation and disease progression in autosomal dominant polycystic kidney disease.

DIPAK Consortium

BACKGROUND: Prostaglandin E2 (PGE2) plays a physiological role in osmoregulation, a process that is affected early in autosomal dominant polycystic kidney disease (ADPKD). PGE2 has also been implicated in the pathogenesis of ADPKD in pre-clinical models, but human data are limited. Here, we hypothesized that urinary PGE2 excretion is associated with impaired osmoregulation, disease severity and disease progression in patients with ADPKD.
METHODS: Urinary excretions of PGE2 and its metabolite (PGEM) were measured in a prospective cohort of patients with ADPKD. Linear regression was used for the cross-sectional analysis of the associations between urinary PGE2 and PGEM excretions, markers of osmoregulation and disease severity. Cox regression and linear mixed models were used for the longitudinal analysis of the associations between urinary PGE2 and PGEM excretions and disease progression. In two separate intervention studies we quantified the effect of starting tolvaptan and adding hydrochlorothiazide to tolvaptan on urinary PGE2 and PGEM excretions.
RESULTS: In 562 patients with ADPKD (61% female, eGFR 63 ± 28 ml/min/1.73 m2) higher urinary PGE2 or PGEM excretions were independently associated with higher plasma copeptin, lower urine osmolality, lower eGFR and greater total kidney volume. Patients with higher baseline urinary PGE2 and PGEM excretions had an increased risk of 40% eGFR loss or kidney failure (HR 1.28 and 1.47 for each doubling in urinary PGE2 or PGEM excretions) and a faster change in eGFR over time (-0.39 and -0.53 ml/min/1.73 m2/year for each doubling in urinary PGE2 or PGEM excretions). In the intervention studies, inducing polyuria by tolvaptan significantly increased urinary PGEM excretion, while reducing polyuria by adding hydrochlorothiazide to tolvaptan significantly increased urinary PGE2 excretion.
CONCLUSIONS: Higher urinary PGE2 and PGEM excretions in patients with ADPKD are associated with parameters of osmoregulation, disease severity and progression.

DOI: https://doi.org/10.2215/CJN.0000000000000269
Prenat Diagn. 2023 Aug 19.

Prenatal diagnosis of polycystic kidney caused by biallelic hypomorphic variants in the PKD1 gene.

Yu Zheng, Lo Wong, Angel Hoi Wan Kwan, Zirui Dong, Ka Yin Kwok, Kwong Wai Choy, Hongzheng Dai, Ye Cao.

Heterozygous loss-of-function variants in the PKD1 gene are commonly associated with adult-onset autosomal dominant polycystic kidney disease (ADPKD), where the formation of renal cysts depends on the dosage of the PKD1 gene. Biallelic null PKD1 variants are not viable, but biallelic hypomorphic variants could lead to early-onset PKD. We report a non-consanguineous Chinese family with recurrent fetal polycystic kidney and negative findings in the coding region of the PKHD1 gene or chromosomal microarray analysis. Trio exome analysis revealed compound heterozygous variants of uncertain significance in the PKD1 gene in the index pregnancy: a novel paternally inherited c.7863 + 5G > C and a maternally inherited c.9739C > T, p.(Arg3247Cys). Segregation analysis through long-range PCR followed by nested PCR and Sanger sequencing confirmed another affected fetus had both variants, while the other two normal siblings and the parents carried either variant. Thus, these two variants, both of which were hypomorphic as opposed to null variants, co-segregated with prenatal onset polycystic kidney disease in this family. Functional studies are needed to further determine the impact of these two variants. Our findings highlight the biallelic inheritance of hypomorphic PKD1 variants causing prenatal onset polycystic kidney disease, which provides a better understanding of phenotype-genotype correlation and valuable information for reproductive counseling.

DOI: https://doi.org/10.1002/pd.6419
J Biol Chem. 2023 Aug 12. pii: S0021-9258(23)02186-5. [Epub ahead of print] 105158

Cleavage fragments of the C-terminal tail of polycystin-1 are regulated by oxidative stress and induce mitochondrial dysfunction.

Hannah Pellegrini, Elizabeth H Sharpe, Guangyi Liu, Eiko Nishiuchi, Nicholas Doerr, Kevin R Kipp, Tiffany Chin, Margaret F Schimmel, Thomas Weimbs.

  Mutations in the gene encoding polycystin-1 (PC1) are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). Cysts in ADPKD exhibit a Warburg-like metabolism characterized by dysfunctional mitochondria and aerobic glycolysis. PC1 is an integral membrane protein with a large extracellular domain, a short C-terminal cytoplasmic tail and shares structural and functional similarities with G protein-coupled receptors (GPCRs). Its exact function remains unclear. The C-terminal cytoplasmic tail of PC1 undergoes proteolytic cleavage, generating soluble fragments that are overexpressed in ADPKD kidneys. The regulation, localization, and function of these fragments is poorly understood. Here, we show that a ∼30kDa cleavage fragment (PC1-p30), comprising the entire C-terminal tail, undergoes rapid proteasomal degradation by a mechanism involving the von Hippel-Lindau tumor suppressor protein (pVHL). PC1-p30 is stabilized by reactive oxygen species (ROS), and the subcellular localization is regulated by ROS in a dose-dependent manner. We found that a second, ∼15kDa fragment (PC1-p15), is generated by caspase cleavage at a conserved site (Asp-4195) on the PC1 C-terminal tail. PC1-p15 is not subject to degradation and constitutively localizes to the mitochondrial matrix. Both cleavage fragments induce mitochondrial fragmentation, and PC1-p15 expression causes impaired fatty acid oxidation and increased lactate production, indicative of a Warburg-like phenotype. Endogenous PC1 tail fragments accumulate in renal cyst-lining cells in a mouse model of PKD. Collectively, these results identify novel mechanisms regarding the regulation and function of PC1, and suggest that C-terminal PC1 fragments may be involved in the mitochondrial and metabolic abnormalities observed in ADPKD.

Keywords:  ADPKD; PC1; Polycystic kidney disease; ROS; metabolism; mitochondria; oxidative stress; polycystin-1

DOI:  https://doi.org/10.1016/j.jbc.2023.105158
J Mol Med (Berl). 2023 Aug 16.

Pkhd1cyli/cyli mice have altered renal Pkhd1 mRNA processing and hormonally sensitive liver disease.

Chaozhe Yang, Naoe Harafuji, Ljubica Caldovic, Weiying Yu, Ravindra Boddu, Surajit Bhattacharya, Hayk Barseghyan, Heather Gordish-Dressman, Oded Foreman, Zsuzsa Bebok, Eva M Eicher, Lisa M Guay-Woodford.

  Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd1cyli/cyli (cyli) mice exhibit a severe liver pathology but lack renal disease. Further analysis revealed that several alternatively spliced Pkhd1 mRNA, all containing exon 48, were expressed in cyli kidneys, but in lower abundance than in wild-type kidneys, suggesting that these transcripts escaped from nonsense-mediated decay (NMD). We identified an AAAAAT motif in exon 48 upstream of the cyli mutation which could enable ribosomal frameshifting, thus potentially allowing production of sufficient amounts of FPC for renoprotection. This mechanism, expressed in a species-specific fashion, may help explain the disparities in the renal phenotype observed between Pkhd1 mutant mice and patients with PKHD1-related disease. KEY MESSAGES: The Pkhd1cyli/cyli mouse expresses cystic liver disease, but no kidney phenotype. Pkhd1 mRNA expression is decreased in cyli liver and kidneys compared to wild-type. Ribosomal frameshifting may be responsible for Pkhd1 mRNA escape from NMD. Pkhd1 mRNA escape from NMD could contribute to the absent kidney phenotype.

Keywords:  ARPKD; Cyli; Hepato-renal fibrocystic disease; Mouse model; Pkhd1

DOI:  https://doi.org/10.1007/s00109-023-02351-2
Proc Natl Acad Sci U S A. 2023 08 22. 120(34): e2302603120

RABL2 promotes the outward transition zone passage of signaling proteins in cilia via ARL3.

Rui-Kai Zhang, Wei-Yue Sun, Yan-Xia Liu, Emma Y Zhang, Zhen-Chuan Fan.

  Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.

Keywords:  ARL3; BBSome; Bardet–Biedl syndrome; RABL2; cilia

DOI:  https://doi.org/10.1073/pnas.2302603120
bioRxiv. 2023 Aug 04. pii: 2023.08.02.551695. [Epub ahead of print]

Ciliary ARL13B prevents obesity in mice.

Tiffany T Terry, Eduardo D Gigante, Coralie M Alexandre, Kathryn M Brewer, Staci E Engle, Xinyu Yue, Nicolas F Berbari, Christian Vaisse, Tamara Caspary.

Cilia are near ubiquitous small, cellular appendages critical for cell-to-cell communication. As such, they are involved in diverse developmental and homeostatic processes, including energy homeostasis. ARL13B is a regulatory GTPase highly enriched in cilia. Mice expressing an engineered ARL13B variant, ARL13B V358A which retains normal biochemical activity, display no detectable ciliary ARL13B. Surprisingly, these mice become obese. Here, we measured body weight, food intake, and blood glucose levels to reveal these mice display hyperphagia and metabolic defects. We showed that ARL13B normally localizes to cilia of neurons in specific brain regions and pancreatic cells but is excluded from these cilia in the Arl13b V358A/V358A model. In addition to its GTPase function, ARL13B acts as a guanine nucleotide exchange factor (GEF) for ARL3. To test whether ARL13B's GEF activity is required to regulate body weight, we analyzed the body weight of mice expressing ARL13B R79Q , a variant that lacks ARL13B GEF activity for ARL3. We found no difference in body weight. Taken together, our results show that ARL13B functions within cilia to control body weight and that this function does not depend on its role as a GEF for ARL3. Controlling the subcellular localization of ARL13B in the engineered mouse model, ARL13B V358A , enables us to define the cilia-specific role of ARL13B in regulating energy homeostasis.

DOI: https://doi.org/10.1101/2023.08.02.551695
bioRxiv. 2023 Aug 02. pii: 2023.07.31.551375. [Epub ahead of print]

The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries.

Gang Fu, Katherine Augspurger, Jason Sakizadeh, Jaimee Reck, Raqual Bower, Douglas Tritschler, Long Gui, Daniela Nicastro, Mary E Porter.

Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion.

DOI: https://doi.org/10.1101/2023.07.31.551375
Front Cell Dev Biol. 2023 ;11 1190258

EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO.

Pablo Barbeito, Raquel Martin-Morales, Adrian Palencia-Campos, Juan Cerrolaza, Celia Rivas-Santos, Leticia Gallego-Colastra, Jose Antonio Caparros-Martin, Carolina Martin-Bravo, Ana Martin-Hurtado, Laura Sánchez-Bellver, Gemma Marfany, Victor L Ruiz-Perez, Francesc R Garcia-Gonzalo.

  Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC's main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2's Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies.

Keywords:  Ellis van Creveld syndrome; Hedgehog signaling; SUMO; Weyers acrofacial dysostosis; cilia; ciliopathy; interactome; ubiquitin

DOI:  https://doi.org/10.3389/fcell.2023.1190258
Cell Death Discov. 2023 Aug 17. 9(1): 299

Diabetes impairs fracture healing through Foxo1 mediated disruption of ciliogenesis.

Zahra Chinipardaz, Gongsheng Yuan, Min Liu, Dana T Graves, Shuying Yang.

Foxo1 upregulation is linked to defective fracture healing under diabetic conditions. Previous studies demonstrated that diabetes upregulates Foxo1 expression and activation and diabetes impairs ciliogenesis resulting in defective fracture repair. However, the mechanism by which diabetes causes cilia loss during fracture healing remains elusive. We report here that streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) dramatically increased Foxo1 expression in femoral fracture calluses, which thereby caused a significant decrease in the expression of IFT80 and primary cilia number. Ablation of Foxo1 in osteoblasts in OSXcretTAFoxo1f/f mice rescued IFT80 expression and ciliogenesis and restored bone formation and mechanical strength in diabetic fracture calluses. In vitro, advanced glycation end products (AGEs) impaired cilia formation in osteoblasts and reduced the production of a mineralizing matrix, which were rescued by Foxo1 deletion. Mechanistically, AGEs increased Foxo1 expression and transcriptional activity to inhibit IFT80 expression causing impaired cilia formation. Thus, our findings demonstrate that diabetes impairs fracture healing through Foxo1 mediated inhibition of ciliary IFT80 expression and primary cilia formation, resulting in impaired osteogenesis. Inhibition of Foxo1 and/or restoration of cilia formation has the potential to promote diabetes-impaired fracture healing.

DOI: https://doi.org/10.1038/s41420-023-01562-3
Res Sq. 2023 Aug 03. pii: rs.3.rs-3058335. [Epub ahead of print]

Hedgehog target genes regulate lipid metabolism to drive basal cell carcinoma and medulloblastoma.

Vikas Daggubati, Akshara Vykunta, Abrar Choudhury, Zulekha Qadeer, Kanish Mirchia, Olivier Saulnier, Naomi Zakimi, Kelly Hines, Michael Paul, Linyu Wang, Natalia Jura, Libin Xu, Jeremy Reiter, Michael Taylor, William Weiss, David Raleigh.

Hedgehog (Hh) signaling is essential for development, homeostasis, and regeneration 1 . Misactivation of the Hh pathway underlies medulloblastoma, the most common malignant brain tumor in children, and basal cell carcinoma (BCC), the most common cancer in the United States 2 . Primary cilia regulate Hh signal transduction 3 , but target genes that drive cell fate decisions in response to ciliary ligands or oncogenic Hh signaling are incompletely understood. Here we define the Hh gene expression program using RNA sequencing of cultured cells treated with ciliary ligands, BCCs from humans, and Hh-associated medulloblastomas from humans and mice (Fig. 1a). To validate our results, we integrate lipidomic mass spectrometry and bacterial metabolite labeling of free sterols with genetic and pharmacologic approaches in cells and mice. Our results reveal novel Hh target genes such as the oxysterol synthase Hsd11β1 and the adipokine Retnla that regulate lipid metabolism to drive cell fate decisions in response to Hh pathway activation. These data provide insights into cellular mechanisms underlying ciliary and oncogenic Hh signaling and elucidate targets to treat Hh-associated cancers.

DOI: https://doi.org/10.21203/rs.3.rs-3058335/v1
J Appl Microbiol. 2023 Aug 18. pii: lxad187. [Epub ahead of print]

Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.

Yunbin Lyu, Hao Luo, Shumao Chai, Ying Zhang, Xinyu Fan, Shaochen Wang, Zhiyang Feng.

   AIMS: To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach.
METHODS AND RESULTS: A Tibetan soil metagenomic library was applied for screening cellulase-active clones by function-based metagenomics. The candidate genes in the active clones were identified through bioinformatic analyses and heterologously expressed using an Escherichia coli system. The recombinant endoglucanases were purified and characterized using enzyme assays to determine their bioactivities, stabilities, substrate specificities, and other enzymatic properties. A novel endoglucanase gene Zfeg1907 was identified, which consisted of a glycoside hydrolase family 44 (GH44) catalytic domain along with a polycystic kidney disease (PKD) domain and a fibronectin type Ⅲ (Fn3) domain at the C terminal. Recombinant enzyme ZFEG1907 and its truncated mutant ZFEG1907t (ΔPKDΔFn3) were successfully expressed and purified. The two recombinants exhibited catalytic activities towards carboxymethyl cellulose, konjac glucomannan and lichenan. Both enzymes had an optimal temperature of 50°C and an optimal pH value of 5.0. The catalytic activities of both recombinant enzymes were promoted by adding Zn2+ and Ca2+ at the final concentration of 10 mM. The Km value of ZFEG1907 were lower while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, konjac glucomannan and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a β-sandwich structure.
CONCLUSIONS: A novel endoglucanase ZFEG1907 contained a GH44 catalytic domain and a PKD-Fn3 domain was characterized. The PKD-Fn3 domains were not indispensable for the activity but contributed to the enzyme binding of the polysaccharide substrates as a carbohydrate-binding module (CBM).

Keywords:  Endoglucanase; Fibronectin type Ⅲ domain; GH44 catalytic domain; Polycystic kidney disease domain; Soil metagenome library

DOI:  https://doi.org/10.1093/jambio/lxad187
FASEB J. 2023 09;37(9): e23138

The putative protein kinase Stk36 is essential for ciliogenesis and CSF flow by associating with Ulk4.

Hongye Zhang, Meimei Yang, Jianhua Zhang, Li Li, Tianyuan Guan, Jiaxin Liu, Xuanwei Gong, Fan Yang, Sanbing Shen, Min Liu, Yongfeng Han.

  Motile cilia lining on the ependymal cells are crucial for cerebrospinal fluid (CSF) flow and its dysfunction is often associated with hydrocephalus. Unc51-like-kinase 4 (Ulk4) was previously linked to CSF flow and motile ciliogenesis in mice, as the hypomorph mutant of Ulk4 (Ulk4tm1a/tm1a ) developed hydrocephalic phenotype resulted from defective ciliogenesis and disturbed ciliary motility, while the underling mechanism is largely obscure. Here, we report that serine/threonine kinase 36 (STK36), a paralog of ULK4, directly interacts with ULK4 and this was demonstrated by yeast two-hybrid (Y2H) in yeast and coimmunoprecipitation (co-IP) assays in HEK293T cells, respectively. The interaction region was confined to their respective N-terminal kinase domain. The hypomorph mutant of Stk36 (Stk36tmE4-/- ) also developed progressive hydrocephalus postnatally and dysfunctional CSF flow, with multiple defects of motile cilia, including reduced ciliary number, disorganized ciliary orientation, defected axonemal structure and inconsistent base body (BB) orientation. Stk36tmE4-/- also disturbed the expression of Foxj1 transcription factor and a range of other ciliogenesis-related genes. All these morphological changes, motile cilia defects and transcriptional dysregulation in the Stk36tmE4-/- are practically copied from that in Ulk4tm1a/tm1a mice. Taken together, we conclude that both Stk36 and Ulk4 are crucial for CSF flow, they cooperate by direct binding with their kinase domain to regulate the Foxj1 transcription factor pathways for ciliogenesis and cilia function, not limited to CSF flow. The underlying molecular mechanism probably conserved in evolution and could be extended to other metazoans.

Keywords:  Stk36; Ulk4; ependymal cells; hydrocephalus; motile cilia; ventriculomegaly

DOI:  https://doi.org/10.1096/fj.202300481R