Journal of Biological Chemistry

Ti, Xiuxiu;Zuo, Hui;Zhao, Guochun;Li, Yuwei;Du, Minghui;Xu, Liwen;Li, Shengnan;Shan, Zhaoliang;Gao, Yuxue;Gan, Guangming;Wang, Yan;Zhang, Qing

doi: 10.1016/j.jbc.2025.110208pmid: N/A

<h2>Abstract</h2><p>Loss of function of <i>parkin</i> leads to the mitochondrial dysfunction, which is closely related to Parkinson's disease. However, the <i>in vivo</i> mechanism is far from clear. One of the dogmas is that impaired Parkin causes dysfunction of mitophagy mediated by Pink1-Parkin axis. The other is that impaired Parkin causes Mfn accumulation which leads to mitochondrial dysfunction. Surprisingly, in <i>Drosophila</i> muscles, as reported, the first dogma is not applicable; for the second dogma, our study suggests that Parkin mediates the mitochondrial dysfunction through modulating mitochondrial morphology, which is determined by synergy of both Marf and mitochondrial protein mRpL18 got from our genome-wide screen, whose RNAi rescues <i>parkin</i> RNAi phenotype. Mechanistically, we found that impaired Parkin upregulated both the transcription and protein levels of mRpL18 dependent on its E3 ligase activity, causing the mRpL18 accumulation outside mitochondria. Consequently, cytosolic accumulated mRpL18 competitively bound Drp1, leading to the reduction of the binding of Drp1 to its receptor Fis1, which finally inhibited the mitochondrial fission and tipped the balance to mitochondrial hyperfusion, thereby affected the mitochondrial function. Taken together, our study suggests that impaired Parkin causes mitochondrial hyperfusion due to two reasons: (1) Parkin defect impairs Pink1-Parkin axis-mediated Marf degradation, which promotes mitochondrial fusion; (2) Parkin defect causes mRpL18 accumulation, which inhibits Drp1/Fis1-mediated mitochondrial fission. These two ways function together to drive Parkin-mediated mitochondrial hyperfusion. Therefore, knockdown of either <i>marf</i> or <i>mRpL18</i> can prevent mitochondrial hyperfusion, leading to the rescue of Parkin defect-triggered fly wing phenotypes. Overall, our study unveils a new facet of how Parkin regulates mitochondrial morphology to affect mitochondrial function, which provides new insights for the understanding and treatment of Parkinson's disease.</p>

Vera, Renzo E.;Fernadez-Barrena, Maite G.;Falero, Jose M.;Kwon, John Y.;Garza, Roberto A.;Sigafoos, Ashley N.;Ross, Matthew D.;Toruner, Merih Deniz;Toruner, Murat;Tolosa, Ezequiel J.;Almada, Luciana L.;Huang, Huocong;Brekken, Rolf A.;Fernandez-Zapico, Martín E.

doi: 10.1016/j.jbc.2025.110311pmid:

Flores-Arena, Cristina;Malekos, Eric;Montano, Christy;Covarrubias, Sergio;Sudek, Lisa;Dempsey, Valory;Huynh, Vuky;Katzman, Sol;Carpenter, Susan

doi: 10.1016/j.jbc.2025.110204pmid: N/A

<h2>Abstract</h2><p>Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 36,000 lncRNAs annotated in the human genome we do not know the function for the majority. Here we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important in viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (<i>RP11-542M13.2</i> hereafter referred to as long noncoding RNA regulator of monocyte proliferation, <i>LNCRMP</i>) and the second is a lncRNA called <i>OLMALINC</i> (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of <i>LNCRMP and OLMALINC</i> from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that <i>LNCRMP</i> regulated proapoptotic pathways while knockdown of <i>OLMALINC</i> impacted genes associated with the cell cycle. Data supports both <i>LNCRMP</i> and <i>OLMALINC</i> functioning <i>in cis</i> to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.</p>

Li, Ran;Hu, Lezhen;Zhou, Runlin;Zhou, Jialu;Yang, Jiale;Wang, Moran;Sun, Jinsheng

doi: 10.1016/j.jbc.2025.110298pmid: N/A

<h2>Abstracts</h2><p>Molting is a characteristic feature of crustaceans, closely associated with their growth. Following the molting process, crustaceans experience an explosive increase in muscle mass; however, the specific mechanisms underlying this rapid growth remain to be fully elucidated. This study aims to analyze the mechanisms of accelerated muscle proliferation from the perspective of sugar metabolism. The relationship between glycolysis and HIF-1α expression in shrimp muscle during different molting stages was investigated, revealing decreased glucose levels and elevated lactate concentrations in the D0-1 subphase. These findings suggest a Warburg-like effect occurring during pre-molting. Notably, HIF-1α expression was consistently higher in the D0-1, D2, and D3 subphases compared to other stages, indicating its pivotal role in muscle cell proliferation and molting regulation. Knockdown of HIF-1α significantly reduced the expression of glycolytic enzymes and inhibited both muscle growth and molting processes. Additionally, this study explored the effects of gill removal on HIF-1α expression; it was found that mechanical injury increased HIF levels, potentially mimicking hypoxic conditions. Specifically, tumor cell-free extracts were observed to enhance the molting rate—likely linked to upregulation of HIF expression. These results imply that such extracts may create a favorable environment for molting by influencing physiological mechanisms associated with HIF activity, thereby facilitating the molting process in shrimp. Overall, these findings suggest a complex interplay between glycolysis, HIF expression, and physiological processes during shrimp molting while highlighting the potential role of HIF as a regulator within these metabolic pathways.</p>

Ma, Guoqiang;Ruan, Xiye;Yang, Bojun;Li, Ningning;Su, Dan;Sun, Shan;Chen, Siqian;Xu, Kangjia;Ying, Zheng;Wang, Hongfeng

doi: 10.1016/j.jbc.2025.110259pmid: N/A

<h2>Abstract</h2><p>Profilin 1 (PFN1) is a key cytoskeletal protein that regulates actin dynamics by incorporating monomeric actin into linear filaments. PFN1 deletion or mutations have been linked to numerous neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the contribution of PFN1 to neurodegenerative pathologies is poorly understood. Recent studies have implicated the role of aberrant cellular membrane-less organelles (MLOs) in neurodegenerative pathogenesis. Here, we demonstrate that PFN1 is involved in the assembly of MLOs, including Cajal bodies and Stress granules. Specifically, depletion of PFN1 leads to abnormal Cajal body accumulation and accelerated maturation into a gel-like state, consequently dysregulating snRNP biogenesis and impairing pre-mRNA splicing efficiency in both neuronal and non-neuronal cells. Similarly, we show that PFN1 knockdown accelerates the assembly of Stress granules in stressed cells. Furthermore, we demonstrate that the ALS-linked PFN1-C71G mutant exhibits a loss of function in the context of MLO biogenesis. We further reveal that the PFN1 deficiency-induced Cajal body dysregulation, but not Stress granule assembly, is caused by cellular actin filament depolymerization. Importantly, the actin filament agonist CN04 rescues Cajal body properties in PFN1-depleted cells. Taken together, our findings shed light on the role of PFN1 in MLO biogenesis and suggest its involvement in neurodegenerative pathogenesis.</p>

Bernhard, Samuel P.;Ruiz, Francesc X.;Remiszewski, Stacy;Todd, Matthew J.;Shenk, Thomas;Kulp, John L.;Chiang, Lillian W.

doi: 10.1016/j.jbc.2025.110274pmid: 40412521

<h2>Abstract</h2><p>Sirtuin 2 (SIRT2) is a ubiquitously expressed cellular enzyme that deacylates protein lysine residues using NAD⁺ as a cofactor. SIRT2-mediated post-translational modifications on a plethora of protein targets position the enzyme to exert a wide-ranging regulatory role in many physiological and pathological processes. More than 39 SIRT2 crystal structures in complex with substrates, products, mimetics of substrates and products, and modulators, have been reported. The Rossmann fold of the catalytic core presents inducible acyl and cofactor binding cavities that accommodate acyl chains of diverse lengths. These structures have provided information for the design of mechanism- and substrate-based inhibitors. Indeed, a specific SIRT2 selectivity pocket has been described and can be targeted by different chemotypes. Despite the determination of many crystal structures, numerous open questions remain, especially relating to the development of small molecule modulators, full or partial activation or inhibition, and relating these effects to different therapeutic applications. Additional questions include understanding the role of the disordered termini, and the role of potential quaternary states (monomer, dimer, and trimer). Deeper insight into these issues may facilitate the development of SIRT2 selective modulators that can be tailored to different pathological scenarios, such as viral infections and cancers, in which either activation or inhibition of SIRT2 may be of therapeutic benefit. This review covers the following topics: (1) primary to quaternary and catalytic structural biology; (2) structural insights into molecular modulation of SIRT2 (inhibition and selectivity by mechanism-based inhibitors, substrate-mimicking inhibitors, C pocket-binding inhibitors, and selectivity pocket binding inhibitors, including insights to activation; and (3) the impact of structural variations (mutations, post-translational modifications, polymorphs, protein interactions). Despite considerable progress, key knowledge gaps remain regarding the design of optimized SIRT2 modulators. Addressing these uncertainties, particularly within the realms of full/partial activation/inhibition, off-target effects, and tailoring modulators to specific pathologies, will require further investigation into the roles of the SIRT2 disordered termini, quaternary states, and post-translational modifications. Ultimately, unraveling these intricacies holds the key to unlocking the therapeutic potential of SIRT2 modulation.</p>

Guy, Adam T.;Kohro, Yuta;Kano, Koki;Huang, Xianyue;Chen, Linchi;Ooashi, Noriko;Inoue, Mariko;Ishii, Nozomi;Yamashita, Tomohiro;Hirabayashi, Yoshio;Imayoshi, Itaru;Matsuo, Ichiro;Greimel, Peter;Tsuda, Makoto;Kamiguchi, Hiroyuki

doi: 10.1016/j.jbc.2025.110324pmid:

Peng, Hui;Lam, Ying-Wai;Lau, Kwok-Fai;Zhou, Zitao;Herdt, Aimee R.;Gelb, Michael H.;Lee, Chris W.

doi: 10.1016/j.jbc.2025.110315pmid: 40449593

<h2>Abstract</h2><p>Krabbe disease (KD) is an autosomal recessive, demyelinating disorder caused by mutations in the <i>GALC</i> gene. Missense mutation variants (MMVs) account for most pathogenic alleles in patients; however, their mechanistic implications and correlations to clinical phenotype remain unclear. To address these questions, we generated a <i>GALC</i> knockout human oligodendrocytic cell line to conduct a robust <i>GALC</i>-MMVs expression study using a panel of 31 GALC-MMVs. Twenty-six clinically-relevant variants dramatically reduced enzyme activity (92-100%). Notably, residual GALC activity strongly correlated with the age of disease-onset in reported cases (Pearson's r >0.94, P <0.0001), suggesting that enzyme activity resulting from MMV expression in this model may serve as a readout for clinical prognostication. Additionally, we identified p.I562T, a predominant pseudodeficiency variant in the newborn screening programs, synergistically impairs protein function and likely triggers disease-onset when inherited co-allelic with certain MMVs. We also identified MMVs that increased protein retention intracellularly and/or decreased secretion. This quantitative analysis of misfolding characteristics could be valuable for identifying MMVs amenable to pharmacological chaperone therapy. Finally, we observed an inverse correlation between residual GALC activity and endogenous psychosine levels in the MMV panel. Given the importance of psychosine as a biomarker for diagnosis and newborn screening, the psychosine accumulation phenotype in our model highlights its potential use for drug discovery. Overall, this study provides a comprehensive overview of the functional deficits and mis-trafficking caused by GALC-MMVs, deepens our understanding of molecular genetics and genotype-phenotype correlations in KD, and highlights the potential of our platform for genetic and therapeutic applications.</p>

Ononye, Onyekachi;Surendran, Sneha;Battapadi, Tripthi;VanderVere-Carozza, Pamela;Howald, Olivia K.;Kantartzis-Petrides, Athena;Jordan, Matthew R.;Ainembabazi, Diana;Wold, Marc S.;Turchi, John J.;Balakrishnan, Lata

doi: 10.1016/j.jbc.2025.110250pmid: 40389081

<h2>ABSTRACT</h2><p>Replication Protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is vital for various aspects of genome maintenance such as replication, recombination, repair and cell cycle checkpoint activation. Binding of RPA to ssDNA protects it from degradation by cellular nucleases, prevents secondary structure formation and suppresses illegitimate recombination. In our current study, we identified the acetyltransferase p300 to be capable of acetylating the 70kDa subunit of RPA <i>in vitro</i> and within cells. The acetylation status of RPA changes throughout the cell cycle, increasing during the S and G2/M phases, and after UV-induced damage. Furthermore, we were able to specifically identify RPA directly associated with the replication fork during the S phase and UV damage to be acetylated. Based on these observations, we evaluated the impact of lysine acetylation on the biochemical properties of RPA. Investigation of binding properties of RPA revealed that acetylation of RPA increased its binding affinity to ssDNA compared to unmodified RPA. The improvement in binding efficiency was a function of DNA length with the greatest increases observed on shorter length ssDNA oligomers. Enzymatic assays further revealed that upon acetylation RPA governs the switch between the short and long flap pathway for Okazaki fragment processing. Our findings demonstrate that p300-dependent, site-specific acetylation enhances RPA's DNA binding properties, potentially regulating its function during various DNA transactions.</p>

Ali, Saima;Ren, Songyang;Agsaoa, Alexis;Mir, Sheema;Mir, Mohammad A.

doi: 10.1016/j.jbc.2025.110173pmid: 40328362

<h2>Abstract</h2><p>Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne nairovirus in the Bunyavirales order. Unlike many viral infections, CCHFV does not induce a host translation shutdown, posing the question of how its mRNAs are efficiently translated amidst competing host transcripts. Here, we show that the CCHFV nucleocapsid protein (N protein) enhances the translation of luciferase reporter mRNA with the help of the viral S-segment mRNA-derived 5' UTR. Chemical inhibition of eIF4E did not affect the N protein-mediated preferential translation of the reporter mRNA. However, translation shutdowns caused by either proteolytic cleavage of eIF4G or chemical inhibition of eIF4A abolished the N protein-mediated preferential translation of the reporter mRNA. These findings demonstrate that the CCHFV N protein requires both eIF4A and eIF4G to facilitate mRNA translation with the assistance of the viral mRNA 5' UTR. Randomization of the viral 5' UTR significantly reduced the translation efficiency of viral S-segment mRNA in cells. Our results demonstrate that wild type S-segment mRNA was heavily engaged with ribosomes, and N protein likely remained associated with the wild type 5' UTR, continuously facilitating ribosome loading, promoting polysome formation, and enhancing protein production. In contrast, most S-segment mRNA with a randomized 5' UTR was largely free from ribosome engagement, explaining the lower protein production from this transcript. Our results demonstrate that the N protein binds to eIF4A and likely reserves a population of eIF4A-eIF4G complexes that remain dedicated to selectively boost the translation of viral S-segment mRNA, thus avoiding competition from host cell transcripts for the same translation machinery.</p>