Synthesis Of Polypeptide Chains Research Articles

From Alpha to Beta – a co-translational way to fold?

ABSTRACT Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion.

DIAGNOSIS AND MANAGEMENT OF THALASEMIC CARDIOMYOPATHY

Thalassemia is a autosomal recessive inherited conditions characterized by decreased or absent polypeptide chain synthesis resulting in reduced hemoglobin in red blood cells and anemia. Synthesis disorders are characterized by ineffective formation of erythropoiesis which causes the need for lifelong transfusions, this will lead to increased intestinal iron absorption and leading to iron accumulation in vital organs, accumulation of iron in the heart can cause myocardial dysfunction including serious complications such as cardiomipopathy of the heart, where thalassemic cardiomyopathy is a the main cause of morbidity was 63.6% and the main cause of death was 71% of thalassemia patients. It is necessary to early detection for thalassemia cardiomyopathy, such as periodic electrocardiography and echocardiography examinations to assess diastolic and systolic functions so that they do not develop into cardiomyopathy. The proper management of heart failure due to thalassemia cardiomyopathy must be optimally given to patients including chelation therapy which can be used as cardioprotective in thalassemic patients.

HAEMATOLOGICAL AND BIOCHEMICAL EFFECTS OF TRANSFUSION OF STORED BLOOD IN TRANSFUSION-DEPENDENT THALASSEMIA PATIENTS

Background: Thalassemia is a genetic disease in which there is an imbalance in the synthesis of globin polypeptide chains. The current study aimed to determine the haematological and biochemical effects of transfusion of seven days stored blood in transfusion-dependent thalassemia patients. Methods: A quasi-experimental study was conducted between January and July 2021 at Muhammad College of Medicine, Peshawar. A total of 20 transfusion-dependent thalassemia patients were selected. The impact of transfusion of 7-days old blood on haemoglobin levels, serum LDH, serum electrolytes, serum-free haemoglobin, serum bilirubin, serum-free iron, serum ferritin, and C-reactive protein were measured. Variations in pre-transfusion and post-transfusion samples were determined using paired-samples t-test, and p<0.05 was considered significant. Results: There was a non-significant difference in increase of haemoglobin levels (p=0.543) after transfusion of fresh and stored blood. Similarly, RBC counts, MCV, MCH, MCHC showed a slightly lower increase as compared to fresh blood. No differences were seen in platelet count between the two groups. However, the rise in white cells was significantly higher after transfusion of 7-days stored blood as compared to fresh blood (p=0.002). A non-significant increase in post-transfusion LDH (p=0.13), direct bilirubin (p=0.76) and indirect bilirubin (p=0.45) was seen. No differences in creatinine, glucose, and uric acid variations were found. Levels of C-reactive protein showed a significantly higher raise when 7-days stored blood was transfused in comparison with fresh blood (p=0.012). Conclusion: At least seven days stored blood can be safely transfused to transfusion-dependent thalassemia patients.

Редкий случай гиподисфибриногенемии у пациента без гематомезенхимальной дисплазии

Фибриноген и коллаген являются важнейшими протеинами межклеточного матрикса, оказывающие прямое влияние как на дифференцировку мезенхимальных стволовых клеток, так и на остеогенез. Кроме того, в клинической практике встречаются случаи протекания данного синдрома не в классическом виде, которые требуют более углубленного изучения звеньев патогенеза и разработки оптимальной коррекции заболевания. Нами проведен поиск литературных данных по ключевым словам в базах данных PubMed, UpToDate, BMJ. Представлен обзор современных научных данных и результаты наблюдения редкого клинического случая у пациента с гиподисфибриногенемией с проявлениями асептического некроза головок бедренных костей без гематомезенхимальной дисплазии. Заключение: В целом, в настоящее время подробно изучены различные аспекты клинико-биохимического патогенеза аномалий фибриногена, но остаются «белые пятна» в изучении тонких патогенетических механизмов на молекулярно-генетическом уровне, а именно: катаболизм белка, реакции раздельного синтеза отдельных полипептидных цепей фибриногена в разных тканях. Остается нерешенным вопрос о целесообразности применения корригирующих гемостаз препаратов таргетного действия при отсутствии у пациента выраженных тромботических и геморрагических проявлений. Fibrinogen and collagen are the most important intercellular matrix proteins that directly effect on the differentiation of mesenchymal stem cells and osteogenesis. In addition, there are non-classical cases of this syndrome that require indepth study of pathogenesis and optimal disease correction. We examined literature data by keywords in databases PubMed, UpToDate, BMJ. We presented a review of current scientific data and observation results of a rare clinical case in a patient with hypodysfibrinogenemia without hematomesenchymal dysplasia with manifestations of aseptic necrosis of femoral heads. Conclusions: In general, various aspects of clinical and biochemical pathogenesis of fibrinogen abnormalities are currently studied in detail, but there are “white spots” in the study of sophisticated pathogenetic mechanisms at the molecular genetic level, namely protein catabolism, the reaction of separate synthesis of individual fibrinogen polypeptide chains in different tissues. Unresolved is the question of applicability of hemostatic corrective drugs in the absence of expressed thrombotic and hemorrhagic manifestations.

Mitochondrial stress-dependent regulation of cellular protein synthesis.

The production of newly synthesized proteins is vital for all cellular functions and is a determinant of cell growth and proliferation. The synthesis of polypeptide chains from mRNA molecules requires sophisticated machineries and mechanisms that need to be tightly regulated, and adjustable to current needs of the cell. Failures in the regulation of translation contribute to the loss of protein homeostasis, which can have deleterious effects on cellular function and organismal health. Unsurprisingly, the regulation of translation appears to be a crucial element in stress response mechanisms. This review provides an overview of mechanisms that modulate cytosolic protein synthesis upon cellular stress, with a focus on the attenuation of translation in response to mitochondrial stress. We then highlight links between mitochondrion-derived reactive oxygen species and the attenuation of reversible cytosolic translation through the oxidation of ribosomal proteins at their cysteine residues. We also discuss emerging concepts of how cellular mechanisms to stress are adapted, including the existence of alternative ribosomes and stress granules, and the regulation of co-translational import upon organelle stress.

Quantifying Single mRNA Translation Kinetics in Living Cells.

One of the last hurdles to quantifying the full central dogma of molecular biology in living cells with single-molecule resolution has been the imaging of single messenger RNA (mRNA) translation. Here we describe how recent advances in protein tagging and imaging technologies are being used to precisely visualize and quantify the synthesis of nascent polypeptide chains from single mRNA in living cells. We focus on recent applications of repeat-epitope tags and describe how they enable quantification of single mRNA ribosomal densities, translation initiation and elongation rates, and translation site mobility and higher-order structure. Together with complementary live-cell assays to monitor translation using fast-maturing fluorophores and mRNA-binding protein knockoff, single-molecule studies are beginning to uncover striking and unexpected heterogeneity in gene expression at the level of translation.

Heat-Shock Proteins MoSsb1, MoSsz1, and MoZuo1 Attenuate MoMkk1-Mediated Cell-Wall Integrity Signaling and Are Important for Growth and Pathogenicity of Magnaporthe oryzae.

The mitogen-activated protein kinase (MAPK) MoMkk1 governs the cell-wall integrity (CWI) pathway in rice blast fungus Magnaporthe oryzae. To understand the underlying mechanism, we have identified MoSsb1 as one of the MoMkk1-interacting proteins. MoSsb1 is a stress-seventy subfamily B (Ssb) protein homolog, sharing high amino acid sequence homology with the 70-kDa heat shock proteins (Hsp70s). Hsp70 are a family of conserved and ubiquitously expressed chaperones that regulate protein biogenesis by promoting protein folding, preventing protein aggregation, and controlling protein degradation. We found that MoSsb1 regulates the synthesis of nascent polypeptide chains and this regulation is achieved by being in complex with other members of Hsp70s MoSsz1 and 40-kDa Hsp40 MoZuo1. MoSsb1 is important for the growth, conidiation, and full virulence of the blast fungus and this role is also shared by MoSsz1 and MoZuo1. Importantly, MoSsb1, MoSsz1, and MoZuo1 are all involved in the regulation of the CWI MAPK pathway by modulating MoMkk1 biosynthesis. Our studies reveal novel insights into how MoSsb1, MoSsz1, and MoZuo1 affect CWI signaling that is involved in regulating growth, differentiation, and virulence of M. oryzae and highlight the conserved functional mechanisms of heat-shock proteins in pathogenic fungi.

The Ribosome as an Allosterically Regulated Molecular Machine.

The ribosome as a complex molecular machine undergoes significant conformational rearrangements during the synthesis of polypeptide chains of proteins. In this review, information obtained using various experimental methods on the internal consistency of such rearrangements is discussed. It is demonstrated that allosteric regulation involves all the main stages of the operation of the ribosome and connects functional elements remote by tens and even hundreds of angstroms. Data obtained using Förster resonance energy transfer (FRET) show that translocation is controlled in general by internal mechanisms of the ribosome, and not by the position of the ligands. Chemical probing data revealed the relationship of such remote sites as the decoding, peptidyl transferase, and GTPase centers of the ribosome. Nevertheless, despite the large amount of experimental data accumulated to date, many details and mechanisms of these phenomena are still not understood. Analysis of these data demonstrates that the development of new approaches is necessary for deciphering the mechanisms of allosteric regulation of the operation of the ribosome.

Thalassemia Spectrum and Prenatal Diagnosis among Voluntary Couples in Shushtar City, during a Five Year Period

Background: Thalassemia and other hemoglobinopathies are inherited disorders that result from genetic defects causing deficient synthesis of hemoglobin polypeptide chains. This study was done to determine the prevalence and to study the complete spectrum of alpha and beta thalassemia gene mutations in the voluntary hemoglobinopathy carrier couples-to-be referred to health centers of Shoushtar city, located in the north Khuzestan province. Methods: Subjects referred to health centers of the city of Shoushtar during 2011 - 2016 were evaluated for the presence of thalassemia minor. After that, alpha and beta thalassemia gene mutations were explored in suspected couples. The samples that underwent PND and diagnosing of causative mutational genes by chorionic villus sampling (CVS) through amniocentesis plus direct or indirect genetic testing at first trimester of pregnancy were proved to be a carrier for alpha thalassemia, beta thalassemia, or other hemoglobinapathies. Results: The mutations for 111/165 (67%) of beta thalassemia chromosomes (19 different mutations) were identified. Of which, IVSII-1 (G > A) and CD 36 - 37 (-T) mutations showed the highest frequency (23.4 for each one) followed by IVSI-110 (G > A) (10%), CD 82-83 (-G) (9%), and -110 (C > T) (7.2%). Among the alpha thalassemia subjects (54/165 (33%)), the -α3.7 mutation was the most common (79/6%) determinant identified in the city of Shoushtar. From 48 voluntary carrier couples who underwent PND, 10 (20.8%) couples were proved to have fetuses with major beta thalassemia; pregnancy termination was done for them. Conclusions: These data suggest that the spectrum of mutations in the city of Shoushtar differs from those reported from other parts of Iran. Therefore, in order to save the time and cost, it is recommended that for the prenatal diagnosis of thalassemia in the city of Shoushtar, analysis of common mutations should be considered as a front line screening strategy. Prenatal diagnosing in this survey demonstrated that beta thalassemia was the most common accounting for all disorders.

Screening for the Known and #913;lpha Thalassemia Deletions by Polymerase Chain Reaction

Thalassemia is the quantitatively inherited disorder of haemoglobin polypeptide chain synthesis. Awareness about the exact frequency of disease in our country, Pakistan, along with the appropriate knowledge of diagnostic and prevention approach will be a step forward to combat thalassemia in Pakistan. The proposed review article will provide updated information regarding the exact frequency and distribution of thalassemia in neonates in Pakistan and Internationally. All this ultimately provide a great help in establishing the policies for thalassemia prevention and control programs in Pakistan. Thalassemia is the quantitatively inherited disorder of hemoglobin polypeptide chain synthesis. Awareness about the exact frequency of disease in our country, Pakistan, along with the appropriate knowledge of diagnostic and prevention approach will be a step forward to combat thalassemia in Pakistan. The proposed review article will provide updated information regarding the exact frequency and distribution of thalassemia in neonates in Pakistan and Internationally. All this ultimately provide a great help in establishing the policies for thalassemia prevention and control programs in Pakistan.

Reasons of Unknown HPLC Peaks during Hemoglobin Analysis for Patients with Beta Thalassemias

Reasons of Unknown HPLC Peaks during Hemoglobin Analysis for Patients with Beta Thalassemias

New Directions for Vectorial Protein Folding

In vivo, protein folding typically proceeds vectorially, from one end of the polypeptide chain to the other. N- to C-terminal vectorial folding occurs during polypeptide chain synthesis by the ribosome, and transport of unfolded polypeptides through the Sec translocon. C- to N-terminal vectorial folding, while less common, occurs during the maturation of some classes of secreted proteins. For example, members of the autotransporter (AT) family of virulence proteins from Gram-negative bacteria are first secreted across the bacterial inner membrane from N- to C-terminus, and then across the outer membrane from C- to N-terminus. Here we demonstrate that these different folding vectors significantly alter the folding mechanism for pertactin, an archetypal AT protein. Moreover, it is now clear that pertactin and other AT proteins exploit these different folding mechanisms to increase secretion efficiency.

Viewing folding of nascent polypeptide chains from ribosomes

Regulation of gene expression occurs at multiple stages and ultimately determines the protein levels in cells. The last decade has witnessed impressive progress in the development of approaches to measuring changes in cellular transcription and protein complement. However, techniques monitoring protein synthesis have lagged far behind. Current proteomic studies gauge the steady state levels of intracellular protein and rely on mass spectrometry, which has a low dynamic range and rarely samples more than 10% of the proteome [1]. RNA-seq is a sensitive approach for assessing transcriptional changes but the transcriptome correlates poorly with changes in protein levels [2,3]. Recent data suggest that the regulation of gene expression within cells occurs predominantly at the level of translation and that protein abundance correlates closely with translation rates [3]. Therefore, monitoring protein synthesis is crucial to understanding cellular homeostasis. Additionally, regulation of protein synthesis at the level of translation permits the cell to respond swiftly to adverse conditions [4]. Therefore, translational control is an integral part of cellular stress response. An intricate process associated with protein synthesis is folding. Proteins must achieve proper tertiary structure to acquire their designated properties and functions. Our current understanding of protein folding is predominantly based on in vitro refolding of denatured full-length proteins [5]. However, under native intracellular conditions, protein folding could occur concurrently with the synthesis of primary polypeptide chains on the ribosomes [6,7]. Cotranslational folding of partially synthesized nascent chains differs from refolding of full length polypeptides owing to the vectorial nature and the relatively slow elongation speed of the translation process. However, it is challenging to capture cotranslational folding events of nascent chains attached to the translating ribosome. Several approaches have been developed to dissect this process but most of them rely on in vitro systems with limited resolution [7,8]. There is thus an urgent need to establish new approaches to monitor cotranslational folding processes in vivo with high resolution. One solution is to look at the folding status of nascent polypeptide chains from the perspective of translating ribosomes.

ChemInform Abstract: Through the Looking Glass —A New World of Proteins Enabled by Chemical Synthesis

AbstractReview: 32 refs.

Detection of Common Deletional Alpha-Thalassemia Spectrum by Molecular Technique in Kelantan, Northeastern Malaysia

Thalassemia is a hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin polypeptide chains. Although thalassemia mostly affects developing countries, there is limited knowledge of its accurate frequency and distribution in these regions. Knowing the prevalence of thalassemia and the frequency of responsible mutations is therefore an important step in the prevention and control program as well as treatment strategies. This study was performed to determine the prevalence and to study the spectrum of gene deletions that are responsible in α-thalassemia in Kelantan, located in northeastern Malaysia. A total 400 first-time blood donors from multiple areas of donation centre were chosen randomly. The presence of three types of α-thalassemia gene deletion in southeast Asian population which were -SEAdeletion, -α 3.7 rightward deletion, and -α 4.2 leftward deletion was detected by using multiplex PCR method. 37 (9.25%) of blood donors were confirmed to have α-thalassemia deletion types. 34 (8%) were heterozygous for α3.7 deletion, 1 (0.25%) was heterozygous for α4.2 deletion, and 2 (0.5%) were heterozygous for SEA type deletion. Alpha-thalassemia-2 with 3.7 deletion was the most common determinant detected in Kelantan Malay compared to other ethnic groups. It has been noted that alpha-thalassemia-2 with 3.7 deletion is the most common type of α-thalassemia throughout the world.

Through the looking glass – a new world of proteins enabled by chemical synthesis

'Chemical ligation'--the regioselective and chemoselective covalent condensation of unprotected peptide segments--has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester-linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high-resolution X-ray crystallography. A 'glycoprotein mimetic' of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. D-protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the D-enantiomers and L-enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear-loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi-racemate with the D-form of the native protein molecule.We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor-A; total synthesis of the mirror-image protein will be used to systematically develop D-protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules.

Synthesis of Poly-l-glutamic Acid Grafted Silica Nanoparticles and Their Assembly into Macroporous Structures

Polypeptide-coated silica nanoparticles represent an interesting class of organic-inorganic hybrids since the ordered secondary structure of the polypeptide grafts imparts functional properties to these nanoparticles. The synthesis of a poly-l-glutamic acid (PLGA) silica nanoparticle hybrid by employing N-carboxyanhydride (NCA) polymerization to synthesize the polypeptide chains and Cu catalyzed azide alkyne cycloaddition reaction to graft these chains onto the silica surface is reported. This methodology enables the synthesis of well-defined polypeptide chains that are attached onto the silica surface at high surface densities. The PLGA-silica conjugate particles are well dispersed in water, and have been thoroughly characterized using multinuclear ((13)C, (29)Si) solid state NMR, thermogravimetric analysis, Fourier transform infrared, dynamic light scattering, and transmission electron microscopy. The pH-dependent reversible aggregation of the PLGA-silica particles, driven by the change in PLGA structure, has also been studied. Preliminary results on the use of aqueous dispersions of silica-PLGA for the preparation of three-dimensional macroporous structures with oriented pores by ice templating methodology are also demonstrated. These macroporous materials, comprising a biocompatible polymer shell covalently attached to rigid inorganic cores, adopts an interesting lamellar structure with fishbone-type architecture.

Birth, life and death of nascent polypeptide chains

The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome (“birth”) to full function (“maturity”) involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation (“death”). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains.

Cell-free synthesis and folding of transmembrane OmpA reveals higher order structures and premature truncations

We use a cell-free transcription–translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain lipids and small vesicles, while lipid rafts do not promote folding compared to the folding in the absence of lipids. Truncated species are observed during translation in the presence of the periplasmic chaperone Skp, which likely binds to the newly synthesized polypeptide chain during cell-free translation and thus prematurely terminate polypeptide chain synthesis. In contrast, folded and unfolded dimers of OmpA correlate negatively with folding yields. This suggests that dimer formation competes with folding and insertion of monomeric OmpA, though folded dimers slowly appear to convert to folded monomers.

Gabapentin: A Stereochemically Constrained γ Amino Acid Residue in Hybrid Peptide Design

Nature has used the all-alpha-polypeptide backbone of proteins to create a remarkable diversity of folded structures. Sequential patterns of 20 distinct amino acids, which differ only in their side chains, determine the shape and form of proteins. Our understanding of these specific secondary structures is over half a century old and is based primarily on the fundamental elements: the Pauling alpha-helix and beta-sheet. Researchers can also generate structural diversity through the synthesis of polypeptide chains containing homologated (omega) amino acid residues, which contain a variable number of backbone atoms. However, incorporating amino acids with more atoms within the backbone introduces additional torsional freedom into the structure, which can complicate the structural analysis. Fortunately, gabapentin (Gpn), a readily available bulk drug, is an achiral beta,beta-disubstituted gamma amino acid residue that contains a cyclohexyl ring at the C(beta) carbon atom, which dramatically limits the range of torsion angles that can be obtained about the flanking C-C bonds. Limiting conformational flexibility also has the desirable effect of increasing peptide crystallinity, which permits unambiguous structural characterization by X-ray diffraction methods. This Account describes studies carried out in our laboratory that establish Gpn as a valuable residue in the design of specifically folded hybrid peptide structures. The insertion of additional atoms into polypeptide backbones facilitates the formation of intramolecular hydrogen bonds whose directionality is opposite to that observed in canonical alpha-peptide helices. If hybrid structures mimic proteins and biologically active peptides, the proteolytic stability conferred by unusual backbones can be a major advantage in the area of medicinal chemistry. We have demonstrated a variety of internally hydrogen-bonded structures in the solid state for Gpn-containing peptides, including the characterization of the C(7) and C(9) hydrogen bonds, which can lead to ribbons in homo-oligomeric sequences. In hybrid alphagamma sequences, distinct C(12) hydrogen-bonded turn structures support formation of peptide helices and hairpins in longer sequences. Some peptides that include the Gpn residue have hydrogen-bond directionality that matches alpha-peptide helices, while others have the opposite directionality. We expect that expansion of the polypeptide backbone will lead to new classes of foldamer structures, which are thus far unknown to the world of alpha-polypeptides. The diversity of internally hydrogen-bonded structures observed in hybrid sequences containing Gpn shows promise for the rational design of novel peptide structures incorporating hybrid backbones.