Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination.

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination.

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD+ binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.

Nonlinear effects of socio-economic factors on urban haze in China: Evidence from spatial econometric smooth transition autoregressive regression approach.

In recent years, China has achieved numerous economic miracles but it has also been plagued by severe air pollution. The frequent hazy weather has severely restricted China's sustainable development. To investigate the nonlinear threshold effect of socio-economic factors on urban haze in China, this study constructs a spatial econometric Smooth Transition Autoregressive Regression (STAR) model based on the STIRPAT theory by using the remote sensing inversion PM2.5 data of 223 prefecture-level and above cities in China mainland during 2004-2016. In this study, the ARAR-STAR model is estimated by quasi-maximum likelihood estimation, and the accuracy of parameter estimation is verified by Monte Carlo simulation, which proves that the ARAR-STAR model constructed in this study is robust. It is concluded that: there is a complex spatial nonlinear relationship between socio-economic factors such as economic development level, population density, advanced industrial structure, energy consumption, opening-up, and haze pollution. The effect of socio-economic factors on haze emission reduction under the spatial influence has complex heterogeneity with the smooth transition between high and low regimes with economic development. The ARAR-STAR model constructed in this paper, which has both individual fixed effects and time fixed effects, expands the form of existing spatial panel nonlinear models and enriches and implements the application of spatial panel smooth transfer threshold models in the environmental field. Not only can it provide policy recommendations for China to achieve "coordinated efficiency in pollution reduction and carbon reduction" as soon as possible, but it also contributes to China's plan to address global climate change and promote global sustainable development.

Design and simulation of a cross-regional collaborative recycling system for secondary resources: A case of lead-acid batteries.

In emerging economies, a significant amount of secondary resources are recycled by the informal sector, which can seriously harm the environment. However, some previous studies of industry management policy design ignored geographical factors. This paper introduces Geographic Information Systems into an agent-based cross-regional recycling model, and employs lead-acid batteries as an example. The model quantitatively displays the evolution of recycling markets in 31 provinces in Mainland China. Results show that: (1) High subsidies can significantly increase the number of formal enterprises in the short term, but their effectiveness decreases when the proportion of government funds in subsidies is above 80% in the long run; (2) The number of illegal recycling enterprises increases by 294% in eight inland provinces (e.g., Ningxia, Xinjiang) when all funds are invested in supervision, but this number is quite small in subsidy policy scenarios; (3) In four eastern regions, including Beijing and Tianjin, the number of illegal recycling enterprises decreases by 84% if supervision is more favored than subsidy; (4) In the optimal case where spatiotemporal factors are considered in all 31 regions, illegal recycling enterprises and waste lead emissions can be reduced by 95.59% and 45.85% nationwide. Our proposed recycling model offers a detailed simulation of multiple regions and diverse stakeholders, and serves as a useful reference for targeted recovery policies. Governments in inland regions like Ningxia and Xinjiang should implement subsidy policies, while supervision policies should be implemented in developed regions like Beijing and Tianjin.

Spectrum of SLC20A2, PDGFRB, PDGFB, and XPR1 mutations in a large cohort of patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.

Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling.

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3',5'-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 Waf1/Cip1 , and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.

Analysis of gene expression and functional characterization of XPR1: a pathogenic gene for primary familial brain calcification.

Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms. Recently, XPR1 variation has accounted for PFBC as another new causative gene. However, little is known about the distribution and basic function of XPR1 and its interaction with the other three pathogenic genes for PFBC (SLC20A2, PDGFRB and PDGFB). The aim of this study was to further clarify the role of XPR1 in PFBC brain pathology. As a result, gene expression profiles showed that XPR1 mRNA was widely expressed throughout the mouse brain. Cerebellum and striatum, most commonly affected in PFBC, contained a higher level of XPR1 protein than other brain regions. Additionally, XPR1 deficiency seriously affected Pi efflux and XPR1 mutations seemed to have an effect through haploinsufficiency mechanism. The immunoprecipitation and immunohistochemical studies demonstrated that XPR1 could interact with PDGFRB and might form a complex on the cell membrane. These results suggested that XPR1 played a fundamental role in the maintenance of cellular phosphate balance in the brain. This provided us with a novel perspective on understanding the pathophysiology of PFBC. The expression networks and interaction with the known pathogenic genes could shed new light on additional candidate genes for PFBC.

Elimination of ALDH+ breast tumor initiating cells by docosahexanoic acid and/or gamma tocotrienol through SHP-1 inhibition of Stat3 signaling.

Study investigated the ability of docosahexaenoic acid (DHA) alone and in combination with gamma-tocotrienol (γT3) to eliminate aldehyde dehydrogenase positive (ALDH+) cells and to inhibit mammosphere formation, biomarker and functional assay for tumor initiating cells (TICs), respectively, in human triple negative breast cancer cells (TNBCs), and investigated possible mechanisms of action. DHA upregulated Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) protein levels and suppressed levels of phosphorylated signal transducer and activator of transcription-3 (pStat3) and its downstream mediators c-Myc, and cyclin D1. siRNA to SHP-1 enhanced the percentage of ALDH+ cells and Stat-3 signaling, as well as inhibited, in part, the ability of DHA to reduce the percentage of ALDH+ cells and Stat-3 signaling. γT3 alone and in combination with DHA reduced ALDH+ TNBCs, up-regulated SHP-1 protein levels, and suppressed Stat-3 signaling. Taken together, data demonstrate the anti-TIC potential of achievable concentrations of DHA alone as well as in combination with γT3.

A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 µM) and its structural analog lenalidomide (10 µM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.

A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades.

Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions.

Digital economy exhibits varying degrees of mitigation of air pollution in China: Total cities-economic subdivisions-urban agglomerations.

Air pollution is a challenge for many cities. The digital economy enhances support for environmental pollution management, while the mechanisms and scaling heterogeneity remain unclear. This study explored the contribution of digital economy development to PM2.5 concentrations control in China and driving mechanisms in different economic subregions and urban agglomerations. Results show that the spillover transfer effect on air pollution mitigation far exceeded the direct effect at different scales. At the national scale, the air pollution mitigation effect of digital economy was mainly through empowering industrial structure optimization and green technology innovation, while it also affected economic subregions and urban agglomerations through varying scenario combinations of pathways with structural optimization, green production, resource allocation, and technology innovation. Research findings provide support for cross-regional joint management strategies of digital economy and air quality and designing regionally differentiated pollution control pathways in the digital economy dimension.

Dynamics of ecosystem services and nonlinear responses to increased anthropogenic pressure.

Escalating global human activities elicit diverse ecosystem service responses, yet understanding remains limited. This study establishes a framework to clarify these responses, focusing on the Yangtze River Economic Belt in China. Analyzing 2000-2020 data, it calculates ecosystem service economic value and human footprint index. It introduces the ecosystem services response index and comprehensive responsiveness index to assess response characteristics and intensity to anthropogenic pressures. Results show a fluctuating decline in ecosystem services and an increase in anthropogenic pressures. There is a nonlinear relationship: ecosystem services decline with rising pressures, following a U-shaped trend. Notably, nonurban agglomerations experience more significant ecosystem service evolution than urban agglomerations due to differing environmental conditions. This highlights regional disparities in human activity impacts on ecosystems, crucial for planning.

Research on the impact and mechanism of financial development on new urbanization: A case study of the Yangtze River Economic Belt.

Based on panel data of 108 cities in China's Yangtze River Economic Belt from 2003 to 2019, a multiple mediation model is used in this study to assess the impact and mechanism of financial development on new urbanization. The main conclusions are that financial development can directly promote the improvement of new urbanization and indirectly improve the level of new urbanization by increasing infrastructure investment, optimizing industrial structure, and enhancing human capital. Further, the financial development of middle-upstream cities has a stronger promoting effect on new urbanization. Whereas the financial development of downstream cities mainly promotes the construction of new urbanization through both infrastructure investment and industrial structure optimization, middle-upstream cities rely more solely on infrastructure investment.

The impact of transportation accessibility on industrial investment in the urban agglomeration around Poyang Lake in China-based on the perspective of ecological security constraints.

Based on the perspective of ecological security constraints, this research takes panel data of 42 counties (cities) in the urban agglomeration around Poyang Lake in China from 2000 to 2020 and uses a spatial econometric model to investigate the impact of transportation accessibility on industrial investment. The findings herein present an obvious spatial relationship between industrial investment among cities under ecological security constraints and reveal how transportation accessibility has a significant spatial effect on industrial investment in this area. Transportation accessibility has promoted industrial investment in the local region but restrained industrial investment in the surrounding areas. A series of endogenous and robustness tests strengthen this conclusion. Lastly, the effect of transportation accessibility on industrial investment in the UAAPYL is influenced by the lake's circle structure and shows obvious heterogeneity.

How does digital trade promote and reallocate the export technology complexity of the manufacturing industry? Evidence from 30 Chinese provinces, 2011-2020.

It is important for China to break the "low-end lock" of the manufacturing value chain worldwide by revealing how digital trade promotes and reallocates the export technology complexity of the manufacturing industry. Panel data for 30 provinces in China from 2011 to 2020 were employed to measure the digital trade development and export technology complexity of the manufacturing industry. Benchmark regression, intermediary effect regression, panel threshold and other models were used to test the promotion and reallocation of digital trade on the export technology complexity of the manufacturing industry. The findings are as follows: (1) Digital trade promotes the export technology complexity of the manufacturing industry, with significant regional heterogeneity (eastern, central and western regions), and the most obvious promotion in technology-intensive manufacturing. (2) Technological innovation and human capital play a reallocation role in the process of digital trade, affecting the technological complexity of manufacturing exports, with mediating effects of 14.19% and 8.61%, respectively. (3) Digital trade promotes and reallocates the export technology complexity of the manufacturing industry through industrial structure upgrading, and a nonlinear relationship was found. These results provide empirical support and a decision-making basis for digital trade in promoting the export technology complexity of the manufacturing industry. The development of digital trade should be encouraged; the differential development of digital trade in the eastern, central, and western regions should be boosted; importance should be attached to the intermediary incentive role of technological innovation and human capital; and the upgrading of the industrial structure should be promoted scientifically.

Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade.

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies. Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

Osteogenic effects of a potent Src-over-Abl-selective kinase inhibitor in the mouse.

Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC(50) 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1-10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 µM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, ß (ß-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by ß-CGRP.

An infinite life cycle assessment model to re-evaluate resource efficiency and environmental impacts of circular economy systems.

Challenges exist in life cycle assessment (LCA) to evaluate resource efficiency and environmental impacts of circular economy systems. Rules attributing recycling benefits/burdens are inconsistent, causing system boundary ambiguity. Besides, LCAs covering one or several life cycles fail to capture the complete resource path, which leads to unfair assessment results for the primary life cycle. This paper develops an infinite life cycle assessment model, which integrates LCA, substance flow analysis, and a state transition matrix into an infinite-life-cycle framework. On this basis, algorithms are formulated to quantify the resource efficiency and attribute environmental impacts following the principle of whole first, then allocation. Our model is demonstrated by a case study of lead-acid batteries. Results show that the resource efficiency of lead in the infinite life cycle assessment model is at least 118.75% higher than that of primary lead derived from the typical finite life cycle models. Measured by the index of environmental toxicity potential, environmental impacts are transferred from the primary product life cycle to recycled product life cycles, with the range fluctuating from 66.26% to 68.12%. Our model enables scholars to make more reasonable assessments for circular economy systems based on traditional LCA adjustment. From the infinite-life-cycle perspective, sustainable production policies should focus on increasing the recycling rate of waste products rather than limiting the exploitation of natural resources.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification.

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.