Peripheral neuropathy as a very rare symptom in a patient with Niemann–Pick type C with negative enzymatic evaluation: a case report

Background Niemann–Pick is a rare metabolic disease distinguished by lysosomal storage defects. This disease is characterized by sphingomyelinase acid deficiency, causing its accumulation in various organs such as the kidneys, spleen, liver, brain, and nerves. Niemann–Pick disease is categorized into four groups: A, B, C, and D. Peripheral neuropathy is an extremely rare complication in patients with Niemann–Pick type C, which certainly leads to neurologic deterioration. Case presentation We report a case of Niemann–Pick type C disease in a 3-year-old Iranian Azeri female patient who was hospitalized twice. The first time was at 1 month of age with symptoms of splenomegaly, jaundice, and elevated liver enzymes, and the second time was at around age 2 for loss of mental and physical abilities. The patient presented with failure to thrive. According to paraclinical examinations, mildly delayed myelination along with a nonspecific periventricular hypersignal intensity was seen. Interestingly, the patient’s Niemann–Pick type C enzymatic function was evaluated twice and was negative on both occasions, while she was positive for NPC1 and NPC2 gene examinations. Conclusions In this study, despite the enzymatic study being negative, Niemann–Pick type C disease was finally confirmed, revealing the importance of mutations in Niemann–Pick type C pathogenesis. Besides, peripheral neuropathy was diagnosed in this patient as a very rare symptom of Niemann–Pick type C.

The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Influence of GSTM1, GSTT1, and GSTP1 genetic polymorphisms on disorders in transplant patients: a systematic review

The glutathione-S-transferase (GST) enzymes are phase II isoenzymes responsible for protection against free radicals and xenobiotics. Since these proteins are described as polymorphic, polymorphisms in genes that encode them may alter enzymatic function and contribute to oxidative stress. In this context, such polymorphisms were already associated with several diseases and multiple therapeutic outcomes. A systematic review was performed to evaluate studies regarding the association between polymorphisms in three genes encoding enzymes of the GST family – GSTM1, GSTT1, and GSTP1 – and disorders in transplant patients. A total of 125 articles on which inclusion and exclusion criteria were applied were identified at PubMed database. Thirty-two studies met the target criteria and were included in the review. The mechanisms by which GST genotypes influence the development of disorders in transplant patients differ by disorder: they may participate in it by decreasing metabolism of drugs administered to patients undergoing transplantation, then exposing them to greater toxicity; by decreasing the repair ability against oxidative stress; or by encoding proteins that may be recognized as foreign, setting of an alloimmune reaction. Although some results are better established – such as GSTM1 null genotype’s role in the development of toxicity events in transplant patients – others require further evidences, as GST influence on the development of pulmonary decline and posttransplant diabetes mellitus (PTDM). The importance of investigating these associations lies in a personalized medicine, in which the high-risk genotype patient has its treatment individualized and its care for prophylaxis and surveillance increased, potentially reducing this population’s morbimortality.

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a $\sim$50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a $\sim$15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1 enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

Association of Mutations Identified in Xanthinuria with the Function and Inhibition Mechanism of Xanthine Oxidoreductase

Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the two-step reaction from hypoxanthine to xanthine and from xanthine to uric acid in purine metabolism. XOR generally carries dehydrogenase activity (XDH) but is converted into an oxidase (XO) under various pathophysiologic conditions. The complex structure and enzymatic function of XOR have been well investigated by mutagenesis studies of mammalian XOR and structural analysis of XOR–inhibitor interactions. Three XOR inhibitors are currently used as hyperuricemia and gout therapeutics but are also expected to have potential effects other than uric acid reduction, such as suppressing XO–generating reactive oxygen species. Isolated XOR deficiency, xanthinuria type I, is a good model of the metabolic effects of XOR inhibitors. It is characterized by hypouricemia, markedly decreased uric acid excretion, and increased serum and urinary xanthine concentrations, with no clinically significant symptoms. The pathogenesis and relationship between mutations and XOR activity in xanthinuria are useful for elucidating the biological role of XOR and the details of the XOR reaction process. In this review, we aim to contribute to the basic science and clinical aspects of XOR by linking the mutations in xanthinuria to structural studies, in order to understand the function and reaction mechanism of XOR in vivo.

Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8. Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs.

ALKBH5 Functions As an Oncogene in Multiple Myeloma

Background: RNA N 6-methyladenosine (m 6A) plays a critical role in regulating gene expression and determining cell fate. The dysregulation of m 6A modulators, including α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5), has been reported to promote tumor development through their enzymatic function. However, the functions of mRNA m 6A and its modulators in multiple myeloma (MM) are largely unknown. Methods: We queried publicly available MM datasets to study the expression profile of m 6A modulators (METTL3, METTL14, WTAP, FTO, and ALKBH5) in MM and their relationships with clinical outcomes in patients with MM. Both gain- and loss-of-function studies were performed to investigate the role of ALKBH5 in MM. The cell proliferation assay, colony formation assay, Annexin V apoptosis analysis, and 5-ethynyl-2′-deoxyuridine (EdU) assay were performed to evaluate the functions of ALKBH5 in MM in vitro. Human MM cell line xenograft models were constructed to examine the effects of ALKBH5 knockdown or overexpression on MM growth in vivo. The rescue assay using catalytically inactive mutant ALKBH5-H204A was conducted to determine whether demethylation activity was required for the function of ALKBH5 in MM. Then, we performed RNA sequencing and m 6A sequencing to explore the key targets that mediated ALKBH5 function in MM. We investigated the gene regulatory mechanism of ALKBH5 in MM by m 6A immunoprecipitation assay, RNA immunoprecipitation assay, RNA decay assay, dual-luciferase reporter assay, and so forth. Gene set enrichment analysis and Western blotting were employed to determine the downstream signaling pathways regulated by ALKBH5 and the recognized target. Results: ALKBH5 was overexpressed in MM and associated with a poor prognosis in patients with MM. The increased ALKBH5 expression was required for the survival and growth of MM cells in vitro and in vivo. Mechanistically, m 6A demethylation activity was required for ALKBH5 to exert tumorigenic effects in MM. Tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) was identified as a functionally important target of ALKBH5. ALKBH5 regulated TRAF1 expression via affecting mRNA stability of TRAF1 in an m 6A- and YTHDF2-dependent manner. ALKBH5 promoted MM cell growth and survival partly through TRAF1-mediated activation of NF-κB and MAPK signaling pathways. Conclusion: Our findings showed that ALKBH5 played an oncogenic role in MM and highlighted that ALKBH5 could potentially be a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

Unveiling the Effect of Low pH on the SARS-CoV-2 Main Protease by Molecular Dynamics Simulations

(1) Background: Main Protease (Mpro) is an attractive therapeutic target that acts in the replication and transcription of the SARS-CoV-2 coronavirus. Mpro is rich in residues exposed to protonation/deprotonation changes which could affect its enzymatic function. This work aimed to explore the effect of the protonation/deprotonation states of Mpro at different pHs using computational techniques. (2) Methods: The different distribution charges were obtained in all the evaluated pHs by the Semi-Grand Canonical Monte Carlo (SGCMC) method. A set of Molecular Dynamics (MD) simulations was performed to consider the different protonation/deprotonation during 250 ns, verifying the structural stability of Mpro at different pHs. (3) Results: The present findings demonstrate that active site residues and residues that allow Mpro dimerisation was not affected by pH changes. However, Mpro substrate-binding residues were altered at low pHs, allowing the increased pocket volume. Additionally, the results of the solvent distribution around Sγ, Hγ, Nδ1 and Hδ1 atoms of the catalytic residues Cys145 and His41 showed a low and high-water affinity at acidic pH, respectively. It which could be crucial in the catalytic mechanism of SARS-CoV-2 Mpro at low pHs. Moreover, we analysed the docking interactions of PF-00835231 from Pfizer in the preclinical phase, which shows excellent affinity with the Mpro at different pHs. (4) Conclusion: Overall, these findings indicate that SARS-CoV-2 Mpro is highly stable at acidic pH conditions, and this inhibitor could have a desirable function at this condition.

Molecular Adaptation to Folivory and the Conservation Implications for Madagascar’s Lemurs

The lemurs of Madagascar include numerous species characterized by folivory across several families. Many extant lemuriform folivores exist in sympatry in Madagascar’s remaining forests. These species avoid feeding competition by adopting different dietary strategies within folivory, reflected in behavioral, morphological, and microbiota diversity across species. These conditions make lemurs an ideal study system for understanding adaptation to leaf-eating. Most folivorous lemurs are also highly endangered. The significance of folivory for conservation outlook is complex. Though generalist folivores may be relatively well equipped to survive habitat disturbance, specialist folivores occupying narrow dietary niches may be less resilient. Characterizing the genetic bases of adaptation to folivory across species and lineages can provide insights into their differential physiology and potential to resist habitat change. We recently reported accelerated genetic change in RNASE1, a gene encoding an enzyme (RNase 1) involved in molecular adaptation in mammalian folivores, including various monkeys and sifakas (genus Propithecus; family Indriidae). Here, we sought to assess whether other lemurs, including phylogenetically and ecologically diverse folivores, might show parallel adaptive change in RNASE1 that could underlie a capacity for efficient folivory. We characterized RNASE1 in 21 lemur species representing all five families and members of the three extant folivorous lineages: (1) bamboo lemurs (family Lemuridae), (2) sportive lemurs (family Lepilemuridae), and (3) indriids (family Indriidae). We found pervasive sequence change in RNASE1 across all indriids, a dN/dS value > 3 in this clade, and evidence for shared change in isoelectric point, indicating altered enzymatic function. Sportive and bamboo lemurs, in contrast, showed more modest sequence change. The greater change in indriids may reflect a shared strategy emphasizing complex gut morphology and microbiota to facilitate folivory. This case study illustrates how genetic analysis may reveal differences in functional traits that could influence species’ ecology and, in turn, their resilience to habitat change. Moreover, our results support the body of work demonstrating that not all primate folivores are built the same and reiterate the need to avoid generalizations about dietary guild in considering conservation outlook, particularly in lemurs where such diversity in folivory has probably led to extensive specialization via niche partitioning.