Identification of ASF1A and HJURP by global H3-H4 histone chaperone analysis as a prognostic two-gene model in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy with poor prognosis. Abnormal expression of H3-H4 histone chaperones has been identified in many cancers and holds promise as a biomarker for diagnosis and prognosis. However, systemic analysis of H3-H4 histone chaperones in HCC is still lacking. Here, we investigated the expression of 19 known H3-H4 histone chaperones in HCC. Integrated analysis of multiple public databases indicated that these chaperones are highly expressed in HCC tumor tissues, which was further verified by immunohistochemistry (IHC) staining in offline samples. Additionally, survival analysis suggested that HCC patients with upregulated H3-H4 histone chaperones have poor prognosis. Using LASSO and Cox regression, we constructed a two-gene model (ASF1A, HJURP) that accurately predicts prognosis in ICGC-LIRI and GEO HCC data, which was further validated in HCC tissue microarrays with follow-up information. GSEA revealed that HCCs in the high-risk group were associated with enhanced cell cycle progression and DNA replication. Intriguingly, HCCs in the high-risk group exhibited increased immune infiltration and sensitivity to immune checkpoint therapy (ICT). In summary, H3-H4 histone chaperones play a critical role in HCC progression, and the two-gene (ASF1A, HJURP) risk model is effective for predicting survival outcomes and sensitivity to immunotherapy for HCC patients.

Cardiac-specific deletion of heat shock protein 60 induces mitochondrial stress and disrupts heart development in mice.

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

Silicon supplementation stabilizes the effect of copper stress, the use of copper chaperones and genes involved: a review.

Heavy metal stress is a major problem in present scenario and the consequences are well known. The agroecosystems are heavily affected by the heavy metal stress and the question arises on the sustainability of the agricultural products. Heavy metals inhibit the process to influence the reactive oxygen species production. When abundantly present copper metal ion has toxic effects which is mitigated by the exogenous application of Si. The role of silicon is to enhance physical parameters as well as gas exchange parameters. Si is likely to increase antioxidant enzymes in response to copper stress which can relocate toxic metals at subcellular level and remove heavy metals from the cell. Silicon regulates phytohormones when excess copper is present. Rate of photosynthesis and mineral absorption is increased in response to metal stress. Silicon manages enzymatic and non-enzymatic activities to balance metal stress condition. Cu transport by the plasma membrane is controlled by a family of proteins called copper transporter present at cell surface. Plants maintain balance in absorption, use and storage for proper copper ion homeostasis. Copper chaperones play vital role in copper ion movement within cells. Prior to that metallochaperones control Cu levels. The genes responsible in copper stress mitigation are discovered in various plant species and their function are decoded. However, detailed molecular mechanism is yet to be studied. This review discusses about the crucial mechanisms of Si-mediated alleviation of copper stress, the role of copper binding proteins in copper homeostasis. Moreover, it also provides a brief information on the genes, their function and regulation of their expression in relevance to Cu abundance in different plant species which will be beneficial for further understanding of the role of silicon in stabilization of copper stress.

Genetic Update and Treatment for Dystonia.

A neurological condition called dystonia results in abnormal, uncontrollable postures or movements because of sporadic or continuous muscular spasms. Several varieties of dystonia can impact people of all ages, leading to severe impairment and a decreased standard of living. The discovery of genes causing variations of single or mixed dystonia has improved our understanding of the disease's etiology. Genetic dystonias are linked to several genes, including pathogenic variations of VPS16, TOR1A, THAP1, GNAL, and ANO3. Diagnosis of dystonia is primarily based on clinical symptoms, which can be challenging due to overlapping symptoms with other neurological conditions, such as Parkinson's disease. This review aims to summarize recent advances in the genetic origins and management of focal dystonia.

Polymorphism in Genes Encoding Adaptor Proteins ST13 and STIP1 and the Risk of Ischemic Stroke: a Pilot Study.

Adaptor proteins stress induced phosphoprotein 1 (STIP1) and ST13 Hsp70 interacting protein (ST13) may play a crucial role in the pathophysiology of ischemic stroke through controlling protein folding, neuronal survival, and regulation of HSP70/HSP90. The present pilot study investigated whether tagSNPs in genes encoding ST13 (rs138335, rs138344, rs7290793, and rs138344) and STIP1 (rs4980524) are associated with ischemic stroke. DNA samples from 721 ischemic stroke patients and 471 healthy controls were genotyped using the MassArray-4. Our research revealed a relationship between rs138344 ST13 and the risk of ischemic stroke, which was seen only in females (risk allele G; OR=1.34, 95%CI=1.07-1.69; p=0.01). The haplotype rs138335G-rs138344C-rs7290793C ST13 was linked with lower risk of ischemic stroke in females: OR=0.42; 95%CI=0.26-0.68; p=0.0005. Thus, ST13 represents a novel genetic marker for ischemic stroke.

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of ß-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management.

In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

The interaction of InvF-RNAP is mediated by the chaperone SicA in Salmonella sp: an in silico prediction.

In this work we carried out an in silico analysis to understand the interaction between InvF-SicA and RNAP in the bacterium Salmonella Typhimurium strain LT2. Structural analysis of InvF allowed the identification of three possible potential cavities for interaction with SicA. This interaction could occur with the structural motif known as tetratricopeptide repeat (TPR) 1 and 2 in the two cavities located in the interface of the InvF and α-CTD of RNAP. Indeed, molecular dynamics simulations showed that SicA stabilizes the Helix-turn-Helix DNA-binding motifs, i.e., maintaining their proper conformation, mainly in the DNA Binding Domain (DBD). Finally, to evaluate the role of amino acids that contribute to protein-protein affinity, an alanine scanning mutagenesis approach, indicated that R177 and R181, located in the DBD motif, caused the greatest changes in binding affinity with α-CTD, suggesting a central role in the stabilization of the complex. However, it seems that the N-terminal region also plays a key role in the protein-protein interaction, especially the amino acid R40, since we observed conformational flexibility in this region allowing it to interact with interface residues. We consider that this analysis opens the possibility to validate experimentally the amino acids involved in protein-protein interactions and explore other regulatory complexes where chaperones are involved.

RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

Role of the Alpha-B-Crystallin Protein in Cardiomyopathic Disease.

Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB, the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.

The parent and family impact of CLN3 disease: an observational survey-based study.

BACKGROUND: CLN3 disease (also known as CLN3 Batten disease or Juvenile Neuronal Ceroid Lipofuscinosis) is a rare pediatric neurodegenerative disorder caused by biallelic mutations in CLN3. While extensive efforts have been undertaken to understand CLN3 disease etiology, pathology, and clinical progression, little is known about the impact of CLN3 disease on parents and caregivers. Here, we investigated CLN3 disease progression, clinical care, and family experiences using semi-structured interviews with 39 parents of individuals with CLN3 disease. Analysis included response categorization by independent observers and quantitative methods. RESULTS: Parents reported patterns of disease progression that aligned with previous reports. Insomnia and thought- and mood-related concerns were reported frequently. "Decline in visual acuity" was the first sign/symptom noticed by n = 28 parents (70%). A minority of parents reported "behavioral issues" (n = 5, 12.5%), "communication issues" (n = 3, 7.5%), "cognitive decline" (n = 1, 2.5%), or "seizures" (n = 1, 2.5%) as the first sign/symptom. The mean time from the first signs or symptoms to a diagnosis of CLN3 disease was 2.8 years (SD = 4.1). Misdiagnosis was common, being reported by n = 24 participants (55.8%). Diagnostic tests and treatments were closely aligned with observed symptoms. Desires for improved or stabilized vision (top therapeutic treatment concern for n = 14, 32.6%), cognition (n = 8, 18.6%), and mobility (n = 3, 7%) dominated parental concerns and wishes for therapeutic correction. Family impacts were common, with n = 34 (81%) of respondents reporting a financial impact on the family and n = 20 (46.5%) reporting marital strain related to the disease. CONCLUSIONS: Collectively, responses demonstrated clear patterns of disease progression, a strong desire for therapies to treat symptoms related to vision and cognition, and a powerful family impact driven by the unrelenting nature of disease progression.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

Defining clinical endpoints in limb girdle muscular dystrophy: a GRASP-LGMD study.

BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders. METHODS/DESIGN: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9). DISCUSSION: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable. TRIAL REGISTRATION: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Elucidation of molecular mechanism of the unfolded protein response.

The endoplasmic reticulum (ER), where newly synthesized secretory and transmembrane proteins are folded and assembled, has the ability to discriminate folded proteins from unfolded proteins and controls the quality of synthesized proteins. Only correctly folded molecules are allowed to move along the secretory pathway, whereas unfolded proteins are retained in the ER.The ER contains a number of molecular chaperones and folding enzymes (ER chaperones hereafter), which assist productive folding of proteins, and therefore newly synthesized proteins usually gain correct tertiary and quaternary structures quite efficiently. Yet unfolded or misfolded proteins even after assistance of ER chaperones are retrotranslocated back to the cytosol, ubiquitinated and degraded by the proteasome. This disposal system is called ER-associated degradation (ERAD). Thus, the quality of proteins in the ER is ensured by two distinct mechanisms, productive folding and ERAD, which have opposite directions.Under a variety of conditions collectively termed ER stress, however, unfolded or misfolded proteins accumulate in the ER, which in turn activates ER stress response or Unfolded Protein Response (UPR). The UPR is mediated by transmembrane proteins in the ER, and three ER stress sensors/transducers, namely IRE1, PERK and ATF6, operates ubiquitously in mammals. Thanks to these signaling pathways, translation is generally attenuated to decrease the burden on the folding machinery; transcription of ER chaperones is induced to augment folding capacity; and transcription of components of ERAD machinery is induced to enhance degradation capacity, leading to maintenance of the homeostasis of the ER. If ER stress sustains, cells undergo to apoptosis.I will talk on the mechanism, evolution, and physiological importance of the UPR and ERAD as well as its involvement in development and progression of various diseases.

HSPB1 as an RNA-binding protein mediates the pathological process of osteoarthritis.

Heat-shock protein beta1 (HSPB1) is a member of the small HSP family, downregulated in osteoarthritis (OA) chondrocytes and demonstrated the capacity to serve as an RNA-binding protein (RBP). This work aimed to explore the profile of HSPB1 bound RNA and reveal the potential regulation mechanism of HSPB1 in OA. In this work, we captured an unbiased HSPB1-RNA interaction map in Hela cells using the iRIP-seq. The results demonstrated that HSPB1 interacted with plentiful of mRNAs and genomic location toward the CDS region. Functional enrichment of HSPB1-related peaks showed the involvement in gene expression, translation initiation, cellular protein metabolic process, and nonsense-mediated decay. HOMER software analysis showed that HSPB1 bound peaks were over-represented in GAGGAG sequences. In addition, ABLIRC and CIMS algorithm indicated that HSPB1 bound to AU-rich motifs and the proportion of AU-rich peaks in 3' UTR were slightly higher than that in other regions. Moreover, HSPB1-binding targets analysis revealed several gens were associated with OA including EGFR, PLEC, COL5A1, and ROR2. The association of OA-related mRNAs to HSPB1 was additionally confirmed in OA tissues by the quantitative RIP-PCR experiments. Further experiment demonstrated the downregulation of HSPB1 in OA tissues. In conclusion, our current study confirmed HSPB1 as an RNA-binding protein and revealed its potential function in the pathological process of OA, providing a reliable insight to further investigate the molecular regulation mechanism of HSPB1 in OA.

A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

Lack of mitochondrial complex I assembly factor NDUFAF2 results in a distinctive infantile-onset brainstem neurodegenerative disease with early lethality.

BACKGROUND: Congenital disorders of the mitochondrial respiratory chain are a heterogeneous group of inborn errors of metabolism. Among them, NADH:ubiquinone oxidoreductase (complex I, CI) deficiency is the most common. Biallelic pathogenic variants in NDUFAF2, encoding the nuclear assembly CI factor NDUFAF2, were initially reported to cause progressive encephalopathy beginning in infancy. Since the initial report in 2005, less than a dozen patients with NDUFAF2-related disease have been reported. METHODS: Clinical, biochemical, and neuroradiological features of four new patients residing in Northern Israel were collected during 2016-2022 at Emek Medical Center. Enzymatic activities of the five respiratory-chain complexes were determined in isolated fibroblast mitochondria by spectrophotometric methods. Western blot analyses were conducted with anti-human NDUFAF2 antibody; antibody against the mitochondrial marker VDAC1 was used as a loading control. Genetic studies were performed by chromosome microarray analysis using Affymetrix CytoScan 750 K arrays. RESULTS: All four patients presented with infantile-onset growth retardation, ophthalmological impairments with nystagmus, strabismus (starting between 5 and 9 months), and further progressed to life-threatening episodes of apnea usually triggered by trivial febrile illnesses (between 10 and 18 months) with gradual loss of acquired developmental milestones (3 of 4 patients). Serial magnetic-resonance imaging studies in two of the four patients showed a progressive pattern of abnormal T2-weighted hyperintense signals involving primarily the brainstem, the upper cervical cord, and later, the basal ganglia and thalami. Magnetic-resonance spectroscopy in one patient showed an increased lactate peak. Disease progression was marked by ventilatory dependency and early lethality. 3 of the 4 patients tested, harbored a homozygous 142-kb partial interstitial deletion that omits exons 2-4 of NDUFAF2. Mitochondrial CI activity was significantly decreased in the only patient tested. Western blot analysis disclosed the absence of NDUFAF2 protein compared to normal controls. In addition, we reviewed all 10 previously reported NDUFAF2-deficient cases to better characterize the disease. CONCLUSIONS: Biallelic loss-of-function mutations in NDUFAF2 result in a distinctive phenotype in the spectrum of Leigh syndrome with clinical and neuroradiological features that are primarily attributed to progressive brainstem damage.

RNA-dependent proteome solubility maintenance in Escherichia coli lysates analysed by quantitative mass spectrometry: Proteomic characterization in terms of isoelectric point, structural disorder, functional hub, and chaperone network.

Protein aggregation, a consequence of misfolding and impaired proteostasis, can lead to cellular malfunctions such as various proteinopathies. The mechanisms protecting proteins from aggregation in complex cellular environments have long been investigated, often from a protein-centric viewpoint. However, our study provides insights into a crucial, yet overlooked actor: RNA. We found that depleting RNAs from Escherichia coli lysates induces global protein aggregation. Our quantitative mass spectrometry analysis identified over 900 statistically significant proteins from the Escherichia coli proteome whose solubility depends on RNAs. Proteome-wide characterization showed that the RNA dependency is particularly enriched among acidic proteins, intrinsically disordered proteins, and structural hub proteins. Moreover, we observed distinct differences in RNA-binding mode and Gene Ontology categories between RNA-dependent acidic and basic proteins. Notably, the solubility of key molecular chaperones [Trigger factor, DnaJ, and GroES] is largely dependent on RNAs, suggesting a yet-to-be-explored hierarchical relationship between RNA-based chaperone (termed as chaperna) and protein-based chaperones, both of which constitute the whole chaperone network. These findings provide new insights into the RNA-centric role in maintaining healthy proteome solubility in vivo, where proteins associate with a variety of RNAs, either stably or transiently.