Biology subjects

Alam, P.

Publications and source records attributed to Alam, P..

22 records · Page 2Linked to original sources

bioRxiv2022

Cryo-EM of prion strains from the same genotype of host identifies conformational determinants

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 [A] resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors (a22L). Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular {beta}-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major {beta}-arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register {beta}-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

biophysics↗

bioRxiv2022

Cytoplasmic membrane thinning observed by interfacial dyes is likely a common effect of bactericidal antibiotics

The lipid membrane is a fundamental part of life. However, the effects of different stresses on membranal integrity and physiology are less understood. Using novel 4-aminophthalimide-based membrane-specific dyes (4AP-Cn: n is carbon chain-length), aided with confocal microscopy, fluorescence spectroscopy, molecular dynamics simulations, and flow cytometry, we have studied stress-mediated changes in E. coli membranes. By exploiting the depth-dependent positioning and subsequent environmental sensitivity of the dyes, we have proposed a measure of antibiotic-induced membrane damage: the fluorescence Peak Maxima Difference (PMD) between 4AP-C9 and 4AP-C13. The ROS-influenced PMD quantifies cytoplasmic membrane thickness and measures sensitivity against most bactericidal antibiotics, depending upon the extent of lipid peroxidation. Importantly, we have verified this observation using antibiotic-sensitive and resistant clinical isolates of E. coli and ESKAPE pathogens like K. pneumoniae and Enterobacter spp. This study underscores the role of antibiotic-induced membrane changes; a critical issue given the increasing global antimicrobial resistance.

microbiology↗

bioRxiv2021

Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

Parkinsons disease is mainly caused by aggregation of -synuclein (-syn) in the brain. Exchange of -syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of -syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of -syn monomers and oligomers across an in vitro BBB model system. Both -syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric -syn in brain endothelial cells, we investigated co-localization of -syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric -syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric -syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric -syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric -syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric -syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies. Significance StatementIn the submitted manuscript, we describe the use of a state-of-the-art porcine blood-brain barrier model based on primary cells to get information about this important issue. We identify several hitherto undescribed cellular pathways to mediate polarized transport of alpha-synuclein. One of these paths we find regulated by Rab7 and can be inhibited by targeting several intracellular proteins such as VPS35, Caveolin1 and Rab7. New knowledge describing brain endothelial intracellular transport systems are highly warranted for identifying new target to alleviate Parkinsons disease. We believe that our findings could be the seed to establish new therapeutic strategies against Parkinsons disease and related synucleinopathies.

cell biology↗

bioRxiv2021

Adipocyte deletion of the RNA binding protein HuR induces cardiac hypertrophy and fibrosis

Adipose tissue continues to gain appreciation for its broad role as an endocrine organ, and disruptions in adipose tissue homeostasis plays a central role in cardiovascular physiology. We have previously shown that expression of the RNA binding protein HuR in adipose tissue mediates energy expenditure, but the potential cardiovascular impacts of this finding have not been explored. We show here that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) is sufficient to induce the spontaneous development of cardiac hypertrophy and fibrosis. Hearts from Adipo-HuR-/- mice have increased left ventricular (LV) ejection fraction, rate of pressure generation, and LV posterior wall thickness that is accompanied by an increase in LV/body weight ratio and hypertrophic gene expression. Furthermore, Adipo-HuR-/- hearts display increased fibrosis by picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene co-expression network analysis (WGCNA) to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and LV mass. RNA-seq results demonstrate a significant increase in pro-inflammatory gene expression in the subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF- and IL-6. WGCNA identified a significant enrichment in inflammation, apoptosis/cell death, and vesicle-mediated transport genes among those whose expression most significantly associated with CVD in Adipo-HuR-/-. In conclusion, we demonstrate that the loss of HuR expression in adipose tissue promotes the development of cardiac hypertrophy and fibrosis, potentially through modulation of inflammation and vesicle-mediated transport in scWAT. NEW AND NOTEWORTHYThis work demonstrates the spontaneous development of cardiac hypertrophy and fibrosis upon adipose tissue-specific deletion of the RNA binding protein HuR that appears to be mechanistically driven by HuR-dependent changes in inflammatory and extracellular vesicle transport mediating genes in the subcutaneous white adipose tissue. These results suggest that loss of HuR expression in adipose tissue in obesity, as demonstrated in mouse and humans by our group and others, may contribute to obesity-mediated CVD.

physiology↗