KDM4B: A promising oncology therapeutic target.

Epigenetic modifications are significant in tumor pathogenesis, wherein the process of histone demethylation is indispensable for regulating gene transcription, apoptosis, DNA replication, and repair of damaged DNA. The lysine demethylases (KDMs) serve an essential role in the aforementioned processes, with particular emphasis on the KDM4 family, also referred to as JMJD2. Multiple studies have underscored the significance of the KDM4 family in the regulation of various biological processes including, but not limited to, the cell cycle, DNA repair mechanisms, signaling pathways, and the progression of tumor formation. Nevertheless, it is imperative to elucidate the underlying mechanism of KDM4B, which belongs to the KDM4 gene family. This review presents a comprehensive examination of the structure, mechanism, and function of KDM4B, as well as a critical analysis of the current body of research pertaining to its involvement in tumorigenesis and development. Furthermore, this review explores the potential therapeutic strategies that specifically target KDM4B.

Adenine nucleotide translocase: Current knowledge in post-translational modifications, regulations and pathological implications for human diseases.

Adenine nucleotide translocases (ANTs) are central to mitochondrial integrity and bioenergetic metabolism. This review aims to integrate the progresses and knowledge on ANTs over the last few years, contributing to a potential implication of ANTs for various diseases. Structures, functions, modifications, regulators and pathological implications of ANTs for human diseases are intensively demonstrated here. ANTs have four isoforms (ANT1-4), responsible for exchanging ATP/ADP, possibly composing of pro-apoptotic mPTP as a major component, and mediating FA-dependent uncoupling of proton efflux. ANT can be modified by methylation, nitrosylation and nitroalkylation, acetylation, glutathionylation, phosphorylation, carbonylation and hydroxynonenal-induced modifications. Compounds, including bongkrekic acid, atractyloside calcium, carbon monoxide, minocycline, 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid, cardiolipin, free long-chain fatty acids, agaric acid, long chain acyl-coenzyme A esters, all have an ability to regulate ANT activities. ANT impairment leads to bioenergetic failure and mitochondrial dysfunction, contributing to pathogenesis of diseases, such as diabetes (deficiency), heart disease (deficiency), Parkinson's disease (reduction), Sengers Syndrome (decrease), cancer (isoform shifting), Alzheimer's Disease (coaggregation with Tau), Progressive External Opthalmoplegia (mutation), and Fascioscapulohumeral muscular dystrophy (overexpression). This review improves the understanding of the mechanism of ANT in pathogenesis of human diseases, and opens a window for novel therapeutic strategies targeted on ANT in diseases.

Carbohydrate-binding module tribes.

At present, 69 families of carbohydrate-binding modules (CBMs) have been isolated by statistically significant differences in the amino acid sequences (primary structures) of their members, with most members of different families showing little if any homology. On the other hand, members of the same family have primary and tertiary (three-dimensional) structures that can be computationally aligned, suggesting that they are descended from common protein ancestors. Members of the large majority of CBM families are ß-sandwiches. This raises the question of whether members of different families are descended from distant common ancestors, and therefore are members of the same tribe. We have attacked this problem by attempting to computationally superimpose tertiary structure representatives of each of the 53 CBM families that have members with known tertiary structures. When successful, we have aligned locations of secondary structure elements and determined root mean square deviations and percentages of similarity between adjacent amino acid residues in structures from similar families. Further criteria leading to tribal membership are amino acid chain lengths and bound ligands. These considerations have led us to assign 27 families to nine tribes. Eight of the tribes have members with ß-sandwich structures, while the ninth is composed of structures with ß-trefoils.

ThYme: a database for thioester-active enzymes.

The ThYme (Thioester-active enzYme; http://www.enzyme.cbirc.iastate.edu) database has been constructed to bring together amino acid sequences and 3D (tertiary) structures of all the enzymes constituting the fatty acid synthesis and polyketide synthesis cycles. These enzymes are active on thioester-containing substrates, specifically those that are parts of the acyl-CoA synthase, acyl-CoA carboxylase, acyl transferase, ketoacyl synthase, ketoacyl reductase, hydroxyacyl dehydratase, enoyl reductase and thioesterase enzyme groups. These groups have been classified into families, members of which are similar in sequences, tertiary structures and catalytic mechanisms, implying common protein ancestry. ThYme is continually updated as sequences and tertiary structures become available.

Structural classification of biotin carboxyl carrier proteins.

We gathered primary and tertiary structures of acyl-CoA carboxylases from public databases, and established that members of their biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains occur in one family each and that members of their carboxyl transferase (CT) domains occur in two families. Protein families have members similar in primary and tertiary structure that probably have descended from the same protein ancestor. The BCCP domains complexed with biotin in acyl and acyl-CoA carboxylases transfer bicarbonate ions from BC domains to CT domains, enabling the latter to carboxylate acyl and acyl-CoA moieties. We separated the BCCP domains into four subfamilies based on more subtle primary structure differences. Members of different BCCP subfamilies often are produced by different types of organisms and are associated with different carboxylases.

Exploratory application of an integrated topic-based curriculum in biochemistry experimental teaching.

Biochemistry, a complicated basic course in health sciences, plays a fundamental role in describing pathogenic mechanism of illness in molecular terms, and is required to be learned for all medical students. Due to various obstacles to biochemistry learning and teaching, there is a pressing issue of curriculum reform to arouse the student's enthusiasm in biochemistry learning. In this study, an integrated topic-based biochemistry training program (TBBTP) combining the traditional lectures, question-based learning and experimental practices, was introduced in biochemistry teaching. Its effectiveness was evaluated through examination and questionnaire analyses. Consequently, we found that TBBTP promoted the dissemination of biochemical knowledge via traditional lecture, designated learning issues and experimental practices, and acquisition of various skills through practical operation, presentation, and independent learning. It motivated students to study biochemistry with intense interest and enthusiasm. This study explored the feasibility of the topic-based biochemistry teaching as a supplement to biochemistry curriculum in medical education and as a mainstream pedagogy in biochemistry lab.

ADP/ATP translocase 1 protects against an α-synuclein-associated neuronal cell damage in Parkinson's disease model.

BACKGROUND: ADP/ATP translocase 1 (ANT1) is involved in the exchange of cytosolic ADP and mitochondrial ATP, and its defection plays an important role in mitochondrial pathogenesis. To reveal an etiological implication of ANT1 for Parkinson's disease (PD), a neurodegenerative disorder, a mouse model treated with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neuroblastoma cell model induced by 1-methyl-4-pehny1-pyridine were utilized in this study. RESULTS: The tissue-specific abundance in ANT1 in mouse brains was accessed using the analysis of Western blot and immunohistochemistry. Down-regulated soluble ANT1 was found to be correlated with PD, and ANT1 was associated with PD pathogenesis via forming protein aggregates with α-synuclein. This finding was confirmed at cellular level using neuroblastoma cell models. ANT1 supplement in neuronal cells revealed the protective roles of ANT1 against cytotoxicity caused by MPP+. Protein interaction assay, coupled with the analysis of LC-MS/MS, silver-stained SDS-PAGE and Western blot against anti-ANT1 antibody respectively, illustrated the interaction of ANT1 with α-synuclein using the expressed α-synuclein as a bite. Additionally, a significant increasing ROSs was detected in the MPP+-treated cells. CONCLUSIONS: This study indicated that ANT1 was a potentially causative factor of PD, and led to neuropathogenic injury via promoting the formation of protein aggregates with α-synuclein. This investigation potentially promotes an innovative understanding of ANT1 on the etiology of PD and provides valuable information on developing potential drug targets in PD treatment or reliable biomarkers in PD prognostication.

Mechanistic insights into the effect of phosphorylation on Ras conformational dynamics and its interactions with cell signaling proteins.

Ras undergoes interconversion between the active GTP-bound state and the inactive GDP-bound state. This GTPase cycle, which controls the activities of Ras, is accelerated by Ras GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (SOS). Oncogenic Ras mutations could affect the GTPase cycle and impair Ras functions. Additionally, Src-induced K-Ras Y32/64 dual phosphorylation has been reported to disrupt GTPase cycle and hinder Ras downstream signaling. However, the underlying mechanisms remain unclear. To address this, we performed molecular dynamics simulations (~30 µs in total) on unphosphorylated and phosphorylated K-Ras4B in GTP- and GDP-bound states, and on their complexes with GTPase cycle regulators (GAP and SOS) and the effector protein Raf. We found that K-Ras4B dual phosphorylation mainly alters the conformation at the nucleotide binding site and creates disorder at the catalytic site, resulting in the enlargement of GDP binding pocket and the retard of Ras-GTP intrinsic hydrolysis. We observed phosphorylation-induced shift in the distribution of Ras-GTP inactive-active sub-states and recognized potential druggable pockets in the phosphorylated Ras-GTP. Moreover, decreased catalytic competence or signal delivery abilities due to reduced binding affinities and/or distorted catalytic conformations of GAP, SOS and Raf were observed. In addition, the allosteric pathway from Ras/Raf interface to the distal Raf L4 loop was compromised by Ras phosphorylation. These results reveal the mechanisms by which phosphorylation influences the intrinsic or GAP/SOS catalyzed transformations between GTP- and GDP-bound states of Ras and its signal transduction to Raf. Our findings project Ras phosphorylation as a target for cancer drug discovery.

HARMONIES: A Hybrid Approach for Microbiome Networks Inference via Exploiting Sparsity.

The human microbiome is a collection of microorganisms. They form complex communities and collectively affect host health. Recently, the advances in next-generation sequencing technology enable the high-throughput profiling of the human microbiome. This calls for a statistical model to construct microbial networks from the microbiome sequencing count data. As microbiome count data are high-dimensional and suffer from uneven sampling depth, over-dispersion, and zero-inflation, these characteristics can bias the network estimation and require specialized analytical tools. Here we propose a general framework, HARMONIES, Hybrid Approach foR MicrobiOme Network Inferences via Exploiting Sparsity, to infer a sparse microbiome network. HARMONIES first utilizes a zero-inflated negative binomial (ZINB) distribution to model the skewness and excess zeros in the microbiome data, as well as incorporates a stochastic process prior for sample-wise normalization. This approach infers a sparse and stable network by imposing non-trivial regularizations based on the Gaussian graphical model. In comprehensive simulation studies, HARMONIES outperformed four other commonly used methods. When using published microbiome data from a colorectal cancer study, it discovered a novel community with disease-enriched bacteria. In summary, HARMONIES is a novel and useful statistical framework for microbiome network inference, and it is available at https://github.com/shuangj00/HARMONIES.

Deficiency of D-alanyl-D-alanine ligase A attenuated cell division and greatly altered the proteome of Mycobacterium smegmatis.

D-Alanyl-D-alanine ligase A (DdlA) catalyses the dimerization of two D-alanines yielding D-alanyl-D-alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti-DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N-Nitrosodimethylamine (NDMA)-dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.

Carboxylic ester hydrolases: Classification and database derived from their primary, secondary, and tertiary structures.

We classified the carboxylic ester hydrolases (CEHs) into families and clans by use of multiple sequence alignments, secondary structure analysis, and tertiary structure superpositions. Our work for the first time has fully established their systematic structural classification. Family members have similar primary, secondary, and tertiary structures, and their active sites and reaction mechanisms are conserved. Families may be gathered into clans by their having similar secondary and tertiary structures, even though primary structures of members of different families are not similar. CEHs were gathered from public databases by use of Basic Local Alignment Search Tool (BLAST) and divided into 91 families, with 36 families being grouped into five clans. Members of one clan have standard α/ß-hydrolase folds, while those of other two clans have similar folds but with different sequences of their ß-strands. The other two clans have members with six-bladed ß-propeller and three-α-helix bundle tertiary structures. Those families not in clans have a large variety of structures or have no members with known structures. At the time of writing, the 91 families contained 321,830 primary structures and 1378 tertiary structures. From these data, we constructed an accessible database: CASTLE (CArboxylic eSTer hydroLasEs, http://www.castle.cbe.iastate.edu).

Structural classification and properties of ketoacyl synthases.

Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and the reactions that they catalyze enhances insights into this enzyme group. KSs fall into five families separated by their characteristic primary structures, each having members with the same catalytic residues, mechanisms, and tertiary structures. KS1 members, overwhelmingly named 3-ketoacyl-ACP synthase III or its variants, are produced predominantly by bacteria. Members of KS2 are mainly produced by plants, and they are usually long-chain fatty acid elongases/condensing enzymes and 3-ketoacyl-CoA synthases. KS3, a very large family, is composed of bacterial and eukaryotic 3-ketoacyl-ACP synthases I and II, often found in multidomain fatty acid and polyketide synthases. Most of the chalcone synthases, stilbene synthases, and naringenin-chalcone synthases in KS4 are from eukaryota. KS5 members are all from eukaryota, most are produced by animals, and they are mainly fatty acid elongases. All families except KS3 are split into subfamilies whose members have statistically significant differences in their primary structures. KS1 through KS4 appear to be part of the same clan. KS sequences, tertiary structures, and family classifications are available on the continuously updated ThYme (Thioester-active enzYme) database.

Thioesterases: a new perspective based on their primary and tertiary structures.

Thioesterases (TEs) are classified into EC 3.1.2.1 through EC 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.-). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester-active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.