Intracellular Folding Research Articles

Evaluating Genetic Alterations in Metastatic Pancreatic Neuroendocrine Tumors with Potential Therapeutic and Prognostic Applications: A Comparative In Silico Study with Metastatic Neuroendocrine Tumors Originating from the Small Intestine and Rectum

Abstract Introduction/Objective Pancreatic neuroendocrine tumors (P-NET) are diverse tumors with slow growth and varying levels of potential malignant behavior. The overall 5-year survival rates for P-NET are approximately 95%, 72%, and 23% if localized, and in cases of regional and distant metastasis respectively. These tumors exhibit a low rate of mutations but show varied genomic abnormalities, contributing to their unpredictable nature. In this study, we aimed to find the genetic alterations observed in metastatic P-NET for their potential therapeutic and prognostic applications and compared these tumors to metastatic neuroendocrine tumors originating from the small intestine (SI-NET) and rectum (R-NET). Methods/Case Report We initially extracted RNA-seq datasets from the GEO database. Next, we analyzed the datasets in six defined groups and differentially expressed genes were obtained by comparison to controls. Either of the groups of P-NET, SI-NET, or R-NET cases were evaluated individually and altogether (primary vs metastatic; 4 groups) and in 2 groups comparing primary/metastatic P-NET cases to primary/metastatic cases of SI-NET plus R- NET. Using a Venn diagram, we isolated the commonality between gene expression profiles. Afterward, we obtained the ontology of genes and signaling pathways from KEGG and Enrichr. Finally, we drew the protein network and evaluated the proteins in GEPIA and TCGA clinical databases. Results (if a Case Study enter NA) A total of 212 cases including 113 P-NET (83 primary and 30 metastatic), 81 SI- NET (44 primary and 37 metastatic), and 18 R-NET (3 primary and 15 metastatic) were selected. We found 8 genes (ORM1, HP, APOA2, HRG, KNG1, ALB, SERPINC1, and AHSG) are mutually markedly upregulated in cases of metastatic P-NET and SI-NET but were not identified in metastatic R-NET. The products of these genes were predominantly involved in complement cascades. CABYR, MYCN, and CALU genes were among the most upregulated genes specifically isolated in the metastatic P-NET that played roles in activating intracellular signaling pathways, transcription, and protein folding. Conclusion The shared upregulated genes in metastatic P-NET and SI-NET may support the relatively better prognosis of these tumors in comparison to the metastatic R-NET due to the activation of the complement cascade (immune system). Furthermore, the identified genetic alterations in the metastatic P-NET cases may have potential therapeutic and prognostic applications for future use.

Small-angle X-ray scattering structural insights into alternative pathway of actin oligomerization associated with inactivated state

In addition to the well-known monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can exist in the so-called inactivated form (I-actin). Hsp70 chaperon, prefoldin, and CCT chaperonin are required to obtain native globular state. In contrast, I-actin is spontaneously formed in the absence of intracellular folding machinery. I-actin can be obtained from G-actin by elimination of divalent ion, incubation in presence of small concentrations of denaturants, and by heat exposure. Since G-actin is a quasi-stationary, thermodynamically unstable form, it can gradually transform into inactivated state in the absence of chelating/denaturating agents or heat exposure, but the transition is much slower. I-actin was shown to associate into oligomers up to the molecular weight of 14–16 G-actin monomers, though the structure of these oligomers remains uncharacterized. This study employs small-angle X-ray scattering to reveal novel insights into the oligomerization process of such spontaneously formed inactivated actin. These oligomers are differentiated from F-actin through comparative analysis, highlighting a unique oligomerization pathway.

Targeting endoplasmic reticulum stress signaling in ovarian cancer therapy.

The endoplasmic reticulum (ER), an organelle present in various eukaryotic cells, is responsible for intracellular protein synthesis, post-translational modification, and folding and transport, as well as the regulation of lipid and steroid metabolism and Ca2+ homeostasis. Hypoxia, nutrient deficiency, and a low pH tumor microenvironment lead to the accumulation of misfolded or unfolded proteins in the ER, thus activating ER stress (ERS) and the unfolded protein response, and resulting in either restoration of cellular homeostasis or cell death. ERS plays a crucial role in cancer oncogenesis, progression, and response to therapies. This article reviews current studies relating ERS to ovarian cancer, the most lethal gynecologic malignancy among women globally, and discusses pharmacological agents and possible targets for therapeutic intervention.

Liver transcriptome analysis revealing response to high-temperature stress in Glyptosternum maculatum (Sisoridae: Siluriformes)

Glyptosternum maculatum (G. maculatum) is a species of endemic fish to the aquatic ecosystem of Tibetan Plateau and has been listed as a critically endangered species in the Red List of China’s Vertebrates. The change in water temperature due to global warming may induce plasticity in G. maculatum to adapt to temperature stress. In this study, we performed transcriptome analysis to characterize the genes and pathways involved in temperature stress response in the liver of G. maculatum. Compared with group A (habitat temperature = 12 °C), changes in expression profiles were more significant in C (habitat temperature = 24 °C) group than that in B (habitat temperature = 4 °C) group. These results suggest the possible lack of reponse to cold stress in G. maculatum. A total of 1247 annotated differentially expressed genes (DEGs) were identified between groups A and C. Based on enrichment analysis, 17 candidate genes with potential roles in heat stress were identified. Additionally, eight important heat-stress-reducing pathways were identified and classified into folding, sorting and degradation, endocrine system, signal transduction, translation, infectious diseases (bacterial), glycan biosynthesis and metabolism, and digestive system. This study also showed that heat stress-regulated DEGs are not only related to intracellular biochemical reactions, metabolism, immune response, and protein folding, but also to the response to heat stress in the liver of G. maculatum. In conclusion, our present results revealed the response of G. maculatum to heat stress at the transcriptional level, which is beneficial for further research and conservation of G. maculatum.

Chemical Synthesis of Bioactive Proteins.

Nature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post-expression modifications-transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

Chemical Synthesis of Bioactive Proteins

AbstractNature has developed a plethora of protein machinery to operate and maintain nearly every task of cellular life. These processes are tightly regulated via post‐expression modifications—transformations that modulate intracellular protein synthesis, folding, and activation. Methods to prepare homogeneously and precisely modified proteins are essential to probe their function and design new bioactive modalities. Synthetic chemistry has contributed remarkably to protein science by allowing the preparation of novel biomacromolecules that are often challenging or impractical to prepare via common biological means. The ability to chemically build and precisely modify proteins has enabled the production of new molecules with novel physicochemical properties and programmed activity for biomedical research, diagnostic, and therapeutic applications. This minireview summarizes recent developments in chemical protein synthesis to produce bioactive proteins, with emphasis on novel analogs with promising in vitro and in vivo activity.

Comparative transcriptomic analysis-based identification of the regulation of foreign proteins with different stabilities expressed in Pichia pastoris.

The industrial yeast Pichia pastoris is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed P. pastoris strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability. Wild-type cC, I66Q and ΔW were each overexpressed in P. pastoris without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in P. pastoris in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed. The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For P. pastoris cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability. These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in P. pastoris.

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

Algal-bacterial intervention as a management tool for next-generation aquaculture sustainability

Emerging occupational and environmental health problems are currently major priorities that need to be tackled along with existing traditional public health problems like communicable diseases, malnutrition, poor environmental sanitation, and inadequate medical facilities. Although toxicologists have expressed deep concern and have prioritized these issues, public awareness, toxicological databases, suitable early diagnosis, specific preventive and therapeutic measures are some of the major issues which requires major attention. Among various strategies which may address these issues include use of modern technologies, amenities, and resources for detecting toxic substances or their derivatives at nanogram (ng) levels.These steps will be important to specifically quantify various toxicants and facilitate future research in order to (i) safe and specific therapeutic or preventive measures and (ii) early diagnosis/ biomarkers to examine effects at the molecular level. It has recently been realized that most of the diseases related to toxicants are incurable; therefore, the best course of action in dealing with them is prevention (Flora, 2008). Heavy metal induced toxicity and its associated complications have become a major issue in the medical world. Heavy metals accumulation in the environment and their associated health hazards still need to be extensively studied as there are many unanswered questions like early diagnosis and specific treatment particularly in case of chronic exposure. Exposure to heavy metals (like lead, cadmium and mercury) or metalloids like arsenic come from a variety of sources, including gasoline, fertilizers, paints, sewage sludge, ground water, wastewater irrigation, pesticides, coal burning residues, domestic and industrial effluents, and petrochemicals. Heavy metals are characteristic representatives of toxic substances which are not biodegradable, enter the food chain, and accumulate in living systems. Increased concentrations and accumulation of Heavy metals can cause severely damaging effects and associated complications in living organisms and can even lead to the death of the organism. Heavy metals toxicity generally lessens energy levels and can severely damage and decrease the function of the brain, kidney, lungs, and liver. Frequent and continuous exposure to heavy metals or metalloids leads to physical, muscular, and neurological degeneration, emulating disorders such as Parkinson's disease, Alzheimer's disease, Wilson's disease, muscular dystrophy, and multiple sclerosis etc. Exposure to lead toxicity causes ionic and oxidative stress conditions in living organisms, occurring due to an imbalance in free radical production and antioxidant levels which normally neutralize or detoxify the reactive intermediates. Antioxidants can protect against free radical mediated damage, providing its reducing equivalents from sulphur groups of cysteine to reactive oxygen species (ROS) and making them stable (Flora et al., 2013). Elevated levels of ROS damages the cells and cellular components, which results in a harassed condition at the cellular level. The ionic mechanism of lead or arsenic toxicity also causes substantial deviations in apoptosis, ionic transportation, cell adhesion, inter- and intra-cellular signalling, protein folding and maturation, the release of neurotransmitters, and enzyme regulation (Pachauri et al., 2013).

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation

How to fix a broken protein: restoring function to mutant human cystathionine β-synthase.

Inborn errors of metabolism (IEM) comprise a large class of recessive genetic diseases involving disorders of cellular metabolism that tend to be caused by missense mutations in which a single incorrect amino acid is substituted in the polypeptide chain. Cystathionine beta-synthase (CBS) deficiency is an example of an IEM that causes large elevations of blood total homocysteine levels, resulting in phenotypes in several tissues. Current treatment strategies involve dietary restriction and vitamin therapy, but these are only partially effective and do not work in all patients. Over 85% of the described mutations in CBS-deficient patients are missense mutations in which the mutant protein fails to fold into an active conformation. The ability of CBS to achieve an active conformation is affected by a variety of intracellular protein networks including the chaperone system and the ubiquitin/proteasome system, collectively referred to as the proteostasis network. Proteostasis modulators are drugs that perturb various aspects of these networks. In this article, we will review the evidence that modulation of the intracellular protein folding environment can be used as a potential therapeutic strategy to treat CBS deficiency and discuss the pros and cons of such a strategy.

T-complex protein 1 subunit zeta-2 (CCT6B) deficiency induces murine teratospermia.

BackgroundThe CCT complex is an important mediator of microtubule assembly and intracellular protein folding. Owing to its high expression in spermatids, CCT knockdown can disrupt spermatogenesis. In the present report, we therefore evaluated the in vivo functionality of the testis-specific CCT complex component CCT6B using a murine knockout model system.MethodsA CRISPR/Cas9 approach was used to generate Cct6b−/− mice, after which candidate gene expression in these animals was evaluated via qPCR and Western blotting. Testicular and epididymal phenotypes were assessed through histological and immunofluorescent staining assays, while a computer-assisted sperm analyzer was employed to assess semen quality.ResultsCct6b−/− mice were successfully generated, and exhibited no differences in development, fertility, appearance, testis weight, or sperm counts relative to control littermates. In addition, no differences in spermatogenesis were detected when comparingCct6b+/+ and Cct6b−/− testes. However, when progressive motility was analyzed, the ratio of normal sperm was significantly decreased in Cct6b−/− male mice, with nuclear base bending being the primary detected abnormality. In addition, slight decreases in Cct4 and Cct7 expression were detected.ConclusionThese data indicated that CCT6B is an important regulator of murine spermatogenesis, with the loss of this protein resulting in CCT complex dysfunction, providing a foundation for further studies.

Impact of Lignocellulose Pretreatment By-Products on S. cerevisiae Strain Ethanol Red Metabolism during Aerobic and An-aerobic Growth.

Understanding the specific response of yeast cells to environmental stress factors is the starting point for selecting the conditions of adaptive culture in order to obtain a yeast line with increased resistance to a given stress factor. The aim of the study was to evaluate the specific cellular response of Saccharomyces cerevisiae strain Ethanol Red to stress caused by toxic by-products generated during the pretreatment of lignocellulose, such as levulinic acid, 5-hydroxymethylfurfural, furfural, ferulic acid, syringaldehyde and vanillin. The presence of 5-hydroxymethylfurfural at the highest analyzed concentration (5704.8 ± 249.3 mg/L) under aerobic conditions induced the overproduction of ergosterol and trehalose. On the other hand, under anaerobic conditions (during the alcoholic fermentation), a decrease in the biosynthesis of these environmental stress indicators was observed. The tested yeast strain was able to completely metabolize 5-hydroxymethylfurfural, furfural, syringaldehyde and vanillin, both under aerobic and anaerobic conditions. Yeast cells reacted to the presence of furan aldehydes by overproducing Hsp60 involved in the control of intracellular protein folding. The results may be helpful in optimizing the process parameters of second-generation ethanol production, in order to reduce the formation and toxic effects of fermentation inhibitors.

The Role of Cyclophilins in Inflammatory Bowel Disease and Colorectal Cancer.

Cyclophilins (Cyps) is a kind of ubiquitous protein family in organisms, which has biological functions such as promoting intracellular protein folding and participating in the pathological processes of inflammation and tumor. Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are two common intestinal diseases, but the etiology and pathogenesis of these two diseases are still unclear. IBD and CRC are closely associated, IBD has always been considered as one of the main risks of CRC. However, the role of Cyps in these two related intestinal diseases is rarely studied and reported. In this review, the expression of CypA, CypB and CypD in IBD, especially ulcerative colitis (UC), and CRC, their relationship with the development of these two intestinal diseases, as well as the possible pathogenesis, were briefly summarized, so as to provide modest reference for clinical researches and treatments in future.

Chaperonin-Containing TCP-1 Promotes Cancer Chemoresistance and Metastasis through the AKT-GSK3β-β-Catenin and XIAP-Survivin Pathways.

Simple SummaryCCT is a chaperonin that participates in folding intracellular proteins. We found that endogenously high expression of the subunit CCT-β is associated with a poorer chemotherapy response in clinical cancer patients. Using two cancer cell lines with higher CCT-β levels, a triple-negative breast cancer cell line MDA-MB-231 and a highly metastatic non-small-cell lung cancer cell line CL1-5, we demonstrated that upregulation of CCT-β expression correlated with chemoresistance and metastasis of these cancer cells in vitro and in vivo. Mechanistic studies allowed us to identify the AKT-GSK3β-β-catenin and XIAP-Survivin pathways promoted by CCT-β to account for the observations. The results provided by our studies are important for developing diagnostic and therapeutic strategies for combating CCT-β-overexpressed cancers.Chaperonin-containing TCP-1 (CCT) is a chaperonin composed of eight subunits that participates in intracellular protein folding. Here, we showed that increased levels of subunits of CCT, particularly CCT-β, were significantly correlated with lower survival rates for cancer patients. Endogenously high expression of CCT-β was found in cancer cell lines, such as the triple-negative breast cancer cell line MDA-MB-231 and the highly metastatic non-small-cell lung cancer cell line CL1-5. Knocking down CCT-β in these cancer cells led to decreased levels of anti-apoptotic proteins, such as XIAP, as well as inhibited phosphorylation of Ser473-AKT and GSK3, resulting in decrease of the nucleus-entering form of β-catenin; these changes reduced the chemoresistance and migration/invasion of the cells. Conversely, overexpression of CCT-β recovered the chemoresistance and cell migration/invasion by promoting the AKT-GSK3β-β-catenin and XIAP-Survivin pathways. Coimmunoprecipitation data revealed that the CCT complex might directly bind and stabilize XIAP and β-catenin. This study not only elucidates the roles of CCT in chemoresistance and metastasis, which are two major obstacles for current cancer therapy, but also provides a possible therapeutic strategy against cancers with overexpressed CCT-β.

TEM and STEM-EDS evaluation of metal nanoparticle encapsulation in GroEL/GroES complexes according to the reaction mechanism of chaperonin

Abstract Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy–energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.

Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

Metabolic changes alter the cellular milieu; can this also change intracellular protein folding? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change protein folding, arguably, it should also alter mutational buffering. Here we find that altered cellular metabolic states in E. coli buffer distinct mutations on model proteins. Buffered-mutants have folding problems in vivo and are differently chaperoned in different metabolic states. Notably, this assistance is dependent upon the metabolites and not on the increase in canonical chaperone machineries. Being able to reconstitute the folding assistance afforded by metabolites in vitro, we propose that changes in metabolite concentrations have the potential to alter protein folding capacity. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in cellular proteostasis.

Regulation of Proteasome Activity by (Post-)transcriptional Mechanisms.

Intracellular protein synthesis, folding, and degradation are tightly controlled processes to ensure proper protein homeostasis. The proteasome is responsible for the degradation of the majority of intracellular proteins, which are often targeted for degradation via polyubiquitination. However, the degradation rate of proteins is also affected by the capacity of proteasomes to recognize and degrade these substrate proteins. This capacity is regulated by a variety of proteasome modulations including (1) changes in complex composition, (2) post-translational modifications, and (3) altered transcription of proteasomal subunits and activators. Various diseases are linked to proteasome modulation and altered proteasome function. A better understanding of these modulations may offer new perspectives for therapeutic intervention. Here we present an overview of these three proteasome modulating mechanisms to give better insight into the diversity of proteasomes.

Association between endoplasmic reticulum stress and risk factors of diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common and challenging ocular complications of diabetes mellitus. As a chronic, progressive ocular disease that poses a serious threat to vision, DR has gradually become a leading cause of blindness worldwide. Emerging evidence points to an important role of endoplasmic reticulum (ER) stress in not only maintaining the steady-state equilibrium in the body, but also in intracellular synthesis, protein folding, and other essential functions. Recent studies have demonstrated clear associations between ER stress-related physiological functions and the pathogenesis of DR. When cells are stimulated by external stimuli, UPR pathway is activated firstly to protect it. However, long-term harmful factors can induce ER stress. which interferes with the physiological metabolism of retinal cells and participates in the occurrence of DR via the ATF6 pathway, PERK pathway and IRE1 pathway. At present, ER stress blocker is expected to become a new anti-DR therapy. Thus, understanding the relationship between ER stress and DR will help to develop new effective preventative treatments. In this review, we summarize the risk factors of DR pathogenesis induced by ER stress toward revealing potentially new therapeutic targets.

P022 Variations in surface expression and stability among human leukocyte antigens (HLA-B)

Aim Among HLA class I (HLA-I) molecules, HLA-B allotypes are the most polymorphic and have dominant influences upon disease outcomes. HLA-B allotypes are known to differ markedly in their intracellular folding and peptidome characteristics. The Aim of this study was to assess whether intracellular assembly or peptidome variations influence the cell surface expression levels or stabilities of HLA-B allotypes. Methods Commercial HLA-Bw4 and HLA-Bw6 antibodies were assessed for HLA-I specificities using Luminex beads, and antibody binding differences were quantified. 244 healthy donors were recruited and HLA genotyping was performed using next generation sequencing (NGS). Donors were selected for further measurements based on heterozygosity for the Bw4 and Bw6 epitopes within their HLA-B antigens, and based on the absence of cross-reactive HLA-A antigens. Using quantitative flow cytometry, antibody binding capacity (ABC) values were measured on freshly isolated lymphocyte subsets using HLA-Bw4, HLA-Bw6 and total class I-specific antibodies. For each selected donor, HLA-I ABC measurements were undertaken at multiple time points. For HLA-B alleles with measurements from at least three donors, averaged ABC values for HLA-Bw4 and HLA-Bw6 were analyzed directly or as a ratio relative to total HLA-I, after taking into account relevant antibody recognition differences. For some HLA-Bw4 and HLA-Bw6 allotypes, cell surface stabilities were also assessed on freshly isolated lymphocytes in the presence of brefeldin A. Results Statistically significant allele-dependent differences are measured in cell surface HLA-B expression levels. For some HLA-B allotypes, lower expression could be related to reduced cell surface stability. Cell surface expression and stability differences appear to be related to the individual peptide-binding characteristics of HLA-B, rather than diversities (promiscuities) of their peptide repertoires. Conclusions Overall, these studies indicate allele-dependent variations in cell surface expression and stability of HLA-B, which in some cases correlate with previously known disease progression outcomes in human immunodeficiency virus (HIV) infections.