Modulation of mitochondria by viral proteins.

Mitochondria are dynamic cellular organelles with diverse functions including energy production, calcium homeostasis, apoptosis, host innate immune signaling, and disease progression. Several viral proteins specifically target mitochondria to subvert host defense as mitochondria stand out as the most suitable target for the invading viruses. They have acquired the capability to control apoptosis, metabolic state, and evade immune responses in host cells, by targeting mitochondria. In this way, the viruses successfully allow the spread of viral progeny and thus the infection. Viruses employ their proteins to alter mitochondrial dynamics and their specific functions by a modulation of membrane potential, reactive oxygen species, calcium homeostasis, and mitochondrial bioenergetics to help them achieve a state of persistent infection. A better understanding of such viral proteins and their impact on mitochondrial forms and functions is the main focus of this review. We also attempt to emphasize the importance of exploring the role of mitochondria in the context of SARS-CoV2 pathogenesis and identify host-virus protein interactions.

Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium.

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Human neurotropic polyomavirus, JC virus, agnoprotein targets mitochondrion and modulates its functions.

JC virus encodes an important regulatory protein, known as Agnoprotein (Agno). We have recently reported Agno's first protein-interactome with its cellular partners revealing that it targets various cellular networks and organelles, including mitochondria. Here, we report further characterization of the functional consequences of its mitochondrial targeting and demonstrated its co-localization with the mitochondrial networks and with the mitochondrial outer membrane. The mitochondrial targeting sequence (MTS) of Agno and its dimerization domain together play major roles in this targeting. Data also showed alterations in various mitochondrial functions in Agno-positive cells; including a significant reduction in mitochondrial membrane potential, respiration rates and ATP production. In contrast, a substantial increase in ROS production and Ca2+ uptake by the mitochondria were also observed. Finally, findings also revealed a significant decrease in viral replication when Agno MTS was deleted, highlighting a role for MTS in the function of Agno during the viral life cycle.

Roles for Autophagy in Esophageal Carcinogenesis: Implications for Improving Patient Outcomes.

Esophageal cancer is among the most aggressive forms of human malignancy with five-year survival rates of <20%. Autophagy is an evolutionarily conserved catabolic process that degrades and recycles damaged organelles and misfolded proteins to maintain cellular homeostasis. While alterations in autophagy have been associated with carcinogenesis across tissues, cell type- and context-dependent roles for autophagy have been reported. Herein, we review the current knowledge related to autophagy in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), the two most common subtypes of esophageal malignancy. We explore roles for autophagy in the development and progression of ESCC and EAC. We then continue to discuss molecular markers of autophagy as they relate to esophageal patient outcomes. Finally, we summarize current literature examining roles for autophagy in ESCC and EAC response to therapy and discuss considerations for the potential use of autophagy inhibitors as experimental therapeutics that may improve patient outcomes in esophageal cancer.

Clinical and translational advances in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most deadly forms of human malignancy characterized by late stage diagnosis, metastasis, therapy resistance and frequent recurrence. Clinical management of ESCC remains challenging and the disease presently lacks approved targeted therapeutics. However, emerging data from recent clinical and translational investigations hold great promise for future progress toward improving patient outcomes in this deadly disease. Here, we review current clinical perspectives in ESCC epidemiology, pathophysiology, and clinical care, highlighting recent advances with potential to impact ESCC prevention, diagnosis and management. We further provide an overview of recent translational investigations contributing to our understanding of the molecular mechanisms underlying ESCC development, progression and therapy response, including insights gained from genetic studies and various murine model systems. Finally, we discuss future perspectives in the clinical and translational realms, along with remaining hurdles that must be overcome to eradicate ESCC.

HIV-1 Nef physically associate with CAMKIIδ - ASK-1 complex to inhibit p38MAPK signalling and apoptosis in infected cells.

Human immunodeficiency type 1 virus accessory protein Nef is a key modulator of AIDS pathogenesis. With no enzymatic activity, Nef regulated functions in host cells largely depends on its ability to form multi-protein complex with the cellular proteins. Here, we identified Calcium (Ca2+)/Calmodulin dependent protein kinase II subunit delta (CAMKIIδ) as novel Nef interacting host protein. Further, we confirmed that Nef mediated [Ca2+]I promote formation of Nef-CAMKIIδ - apoptosis signal-regulating kinase (ASK-1) heterotrimeric complex. The assembly of Nef with CAMKIIδ - ASK-1 inhibits the downstream p38MAPK phosphorylation resulting in abrogation of apoptosis. Further, using competitive peptide inhibitors against Nef binding domains to CAMKIIδ, identified in the present study and ASK-1, individually blocked physical interaction of Nef with CAMKIIδ-ASK-1 complex and restored p38MAPK phosphorylation and apoptosis. Altogether, our study indicates that HIV-Nef modulates cytosolic [Ca2+]I and blocks CAMKIIδ - ASK-1 kinase activity to inhibit apoptosis of infected cells.

HIV-1 Nef and host proteome analysis: Current perspective.

Proteome represents the set of proteins being produced by an organism at a given time. Comparative proteomic profiling of a healthy and diseased state is likely to reflect the dynamics of a disease process. Proteomic techniques are widely used to discover novel biomarkers and decipher mechanisms of HIV-1 pathogenesis. Proteomics is thus emerging as an indispensable tool of monitoring a disease process and intense interactions between HIV-1 and host. Nef is known to regulate various functions in the host to establish the state of infection. This review gives an overview of all proteomic studies done on HIV infection and HIV associated disorders including recent developments in Nef-host proteomic profiling. Here, we propose an emphasis on Nef based proteomic studies. We also discuss the future prospects and the technical and biological challenges involved in proteomic studies. Future studies with Nef related proteomic investigation are likely to identify more targets for diagnosis and therapy.

Identification of MΦ specific POTEE expression: Its role in mTORC2 activation via protein-protein interaction in TAMs.

POTE is known as cancer antigen, expressed in many cancers, along with very few normal tissues like prostate, ovary, testes and embryo. Till date, POTEE identified as majorly expressed POTE paralog. Functionally, POTEF regulates TLR signaling which play important role in innate immunity provided clue about expression of POTE in immune cells. We have chosen three Thp1monocytes, Jurkat T1 and MΦ cells as a model. Here, first time we report expression of POTEE in immune cells specifically only in MΦ but not in monocytes or T-cells. In addition, expression level remains unaltered in MΦ subtypes M1 and M2 and MΦ subjected to various stresses, except MΦs treated with Hyp-CM where MΦs acquires properties of TAMs. In TAMs, POTEE was involved differential protein-protein interaction with mTOR, RICTOR, and Rad51 indicating its biological role in cell invasion through mTORC2 activation. siRNA mediated knockdown of POTEE suggests its importance in cell survival of MΦs as well as TAMs.

HIV-1 Nef-POTEE; A novel interaction modulates macrophage dissemination via mTORC2 signaling pathway.

AIMS: Human immunodeficiency virus -1 [HIV-1] Nef, localizes in different cellular compartments and modulates several cellular pathways. Nef promotes virus pathogenicity through alteration in cell surface receptor expression, apoptosis, protein trafficking etc. Nef regulates viral pathogenesis through interaction with different host proteins. Thus, molecular mechanisms of pathogenesis could be deciphered by identifying novel Nef interacting proteins. MAIN METHODS: HIV-1 Nef interacting proteins were identified by pull down assay and MALDI-TOF analysis. The interaction was further validated through mammalian two hybrid assay. Functional role of this interaction was identified by immunoprecipitation assay, cell invasion and cell migration studies. Fold Change in mRNA levels of CD163, CD206, CCL17 and CCL18 was analyzed using qPCR. KEY FINDINGS: In current study, C. elegans protein ACT4C and its human homolog POTEE was identified to be interacting with Nef. This interaction activates mTORC2 complex, which in-turn activates AKT and PKC-α. The activation of mTORC2 complex was found to be initiated by the interaction of Nef, mTORC2, Rictor to POTEE. The cellular phenotype and functions affected by Nef-POTEE interaction resulted in significant increase in cell invasion and migration of macrophages (MΦ). SIGNIFICANCE: MΦ is primary target of HIV-1 infection where HIV-1 replicates and polarizes immunosuppressive M2 phenotype. Combine effect of M2 phenotype and Viral-host protein interactions compromise the MΦ associated physiological functions. Infected MΦ dissemination into other system also leads to HIV-1 induced malignancies. Therefore, targeting POTEE-Nef interaction can lead to formulating better therapeutic strategy against HIV-1.

HIV-1 Nef CAWLEAQ motif: a regulator of monocytes invasion through ENO1 modulation.

HIV-1 transmission and spread involves significant host-virus interaction. Possible targets for obstacle of HIV-1 lie at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and leads their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. For the viral infectivity and pathogenicity, HIV-1 Nef facilitates immune evasion through protein-protein interactions within host cell. HIV-1 Nef is significant for viral infectivity and pathogenicity. It enhances HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study, HIV-1 Nef forms with specific mutations, revealing sequence variability, were studied for their effects in human SupT1 T cell line and (THP-1) monocyte-macrophage cell line. Proteins being downregulated by Nef in SupT1 were further observed in THP-1, and interestingly two host proteins' (ENO-1 and VDAC1) expression was found to be cell lineage specific, being stimulatory in macrophages/monocytes and inhibitory in T cells. Cell migration, invasion and ADP release studies were employed to determine the biological function affected by Nef-mediated regulation of these two host proteins. ENO1-regulated function: cell invasion was enhanced in THP-1 cells, but was inhibited in SupT1 cells by Nef RP01. In addition, the modulation of proteins and cell invasion remained unaffected by a Nef RP14. These results indicated that regulation of host protein expression and invasive property of host cells by Nef was sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the CAWLEAQ-regulating sequence of Nef. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers were particularly investigated by the peptide. The peptide led to reversal of differential expressions of ENO1 in both SupT1 and THP-1 and inhibition of enhanced invasiveness in THP-1 cells. Further AP-1 was identified as a factor involved in this Nef-mediated regulation of host proteins. Together these findings suggest a possible mechanism of host invasion by HIV-1 through the CAWLEAQ motif of Nef-mediated regulation of ENO1 and identify a potential therapeutic target for HIV-1 entry at mucosal barriers.

Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61) and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.

Rational design and synthesis of novel thiazolidin-4-ones as non-nucleoside HIV-1 reverse transcriptase inhibitors.

A series of novel thiazolidin-4-one analogues, characterized by different substitution patterns at positions C-2 and N-3 of the thiazolidin-4-one scaffold for anti-HIV-1 activity has been investigated. Most of the compounds showed anti-HIV-1 activity at micromolar concentrations when tested in TZM-bl cells in vitro. Among the thirty-three compounds tested, compound 16 was the most potent inhibitor of HIV-1 replication against HIV-1IIIB, HIV-1ADA5, HIV-1UG070 and HIV-1VB59 (EC50=0.02, 0.08, 0.08 and 0.08 µM, respectively) with selectivity index (SI=6940, 1735, 1692 and 1692) against tested viral strains, respectively. The results of the present study suggested that the substitution of the nitro group at 6' position of the C-2 phenyl ring and 4,6-dimethylpyridin-2-yl at the N-3 position of thiazolidin-4-one had a major impact on the anti-HIV-1 activity and was found to lower cytotoxicity. The substitution of the heteroaryl ring with bromo group and bicyclic heteroaryl ring at N-3 thiazolidin-4-one was found to lower anti-HIV-1 activity and increase cytotoxicity. The undertaken docking studies thus facilitated the identification of crucial interactions between the HIV-1 RT enzyme and thiazolidin-4-one inhibitors, which can be used to design new potential inhibitors.

Human beta casein fragment (54-59) modulates M. bovis BCG survival and basic transcription factor 3 (BTF3) expression in THP-1 cell line.

Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human ß -casein fragment (54-59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of M. bovis BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the M. bovis BCG mediated apoptosis resulting in enhanced clearance of M. bovis BCG from THP-1 cell line.

One pot efficient diversity oriented synthesis of polyfunctional styryl thiazolopyrimidines and their bio-evaluation as antimalarial and anti-HIV agents.

An efficient one pot synthesis of a series of pluripotent (E)-1-(3-methyl-5-aryl-7-styryl-5H-thiazolo[3,2-a]pyrimidin-6-yl)-3-arylprop-2-en-1-ones is reported. It involves reaction of 5-acetyl-6-methyl-4-aryl-dihydropyrimidine-2-thiones, propargyl bromide and aromatic aldehydes in presence of ethanolic KOH. The newly synthesized compounds were evaluated for antimalarial activity against Plasmodium falciparum and as HIV-RT inhibitors. Most of the compound displayed potent antimalarial activity with IC(50)<2 µg/mL. Compounds 6, 11 and 20 showed better activity against P. falciparum K1 strains in comparison to standard drug chloroquine. Compounds 6, 11, and 16 exhibited 73.44, 66.92, and 70.81% HIV-RT inhibition at 100 µg/mL.

Lead optimization at C-2 and N-3 positions of thiazolidin-4-ones as HIV-1 non-nucleoside reverse transcriptase inhibitors.

Based on rational drug design approach, a series of novel thiazolidin-4-ones bearing different aryl/heteroaryl moieties at position C-2 and N-3 are synthesized and evaluated as potent inhibitors for human immunodeficiency virus type-1 reverse transcriptase enzyme (HIV-1 RT). An in vitro HIV-1 RT assay showed that the compounds 4, 5, 6, 8, 12, 13, 14 and 17 have shown high inhibition of reverse transcriptase (75.41, 95.50, 98.07, 91.24, 85.27, 77.59, 84.11 & 76.49% inhibition) enzyme activity. Further, cell based assay showed that compounds 4, 5, 8 &12 are identified as the best compounds of the series (EC(50) ranged from 0.09 to 0.8 µg/ml and 0.12 to 1.06 µg/ml) against HIV-1 III(B) and HIV-1 ADA5 strains, respectively. Moreover, the compounds which were active against HIV-1 III(B) and HIV-1 ADA5 were also found to be active against primary isolates (EC(50) ranged from 0.10 to 1.55 µg/ml against HIV-1 UG070 and 0.07 to 1.1 µg/ml against HIV-1 VB59), respectively. Structure-activity relationship (SAR) studies demonstrated the importance of the lipophilic bulky substituent pattern on compact heteroaryl ring at N-3, replacement of C4' at C-2 phenyl by trivalent bioisosteric nitrogen and dihalo groups at C-2 aryl/heteroaryl of thiazolidin-4-ones is crucial for anti-HIV-1 activity. Molecular modeling of compounds 4, 5, 8 and 12 in complex with HIV-1 RT demonstrate that there is good correlation of results obtained from SAR studies. Therefore the compounds 4, 5, 8 and 12 may be considered as good candidates for further optimization of anti-HIV-1 activity.