Leaderless secretory proteins of the neurodegenerative diseases via TNTs: a structure-function perspective.

Neurodegenerative disease-causing proteins such as alpha-synuclein, tau, and huntingtin are known to traverse across cells via exosomes, extracellular vesicles and tunneling nanotubes (TNTs). There seems to be good synergy between exosomes and TNTs in intercellular communication. Interestingly, many of the known major neurodegenerative proteins/proteolytic products are leaderless and are also reported to be secreted out of the cell via unconventional protein secretion. Such classes contain intrinsically disordered proteins and regions (IDRs) within them. The dynamic behavior of these proteins is due to their heterogenic conformations that is exhibited owing to various factors that occur inside the cells. The amino acid sequence along with the chemical modifications has implications on the functional roles of IDRs inside the cells. Proteins that form aggregates resulting in neurodegeneration become resistant to degradation by the processes of autophagy and proteasome system thus leading to Tunneling nanotubes, TNT formation. The proteins that traverse across TNTs may or may not be dependent on the autophagy machinery. It is not yet clear whether the conformation of the protein plays a crucial role in its transport from one cell to another without getting degraded. Although there is some experimental data, there are many grey areas which need to be revisited. This review provides a different perspective on the structural and functional aspects of these leaderless proteins that get secreted outside the cell. In this review, attention has been focused on the characteristic features that lead to aggregation of leaderless secretory proteins (from structural-functional aspect) with special emphasis on TNTs.

Early Bioinformatic Implication of Triacidic Amino Acid Motifs in Autophagy-Dependent Unconventional Secretion of Mammalian Proteins.

Several proteins are secreted outside the cell, and in many cases, they may be identified by a characteristic signal peptide. However, more and more studies point to the evidence for an "unconventional" secretion, where proteins without a hitherto unknown signal are secreted, possibly in conditions of starvation. In this work, we analyse a set of 202 RNA binding mammalian proteins, whose unconventional secretion has recently been established. Analysis of these proteins secreted by LC3 mediation, the largest unconventionally secreted dataset to our knowledge, identifies the role of KKX motif as well as triacidic amino acid motif in unconventional secretion, the latter being an extension of the recent implicated diacidic amino acid motif. Further data analysis evolves a hypothesis on the sequence or structural proximity of the triacidic or KKX motifs to the LC3 interacting region, and a phosphorylatable amino acid such as serine as a statistically significant feature among these unconventionally secreted proteins. This hypothesis, although needs to be validated in experiments that challenge the specific details of each of these aspects, appears to be one of the early steps in defining what may be a plausible signal for unconventional protein secretion.

Identification and in silico analysis of the origin recognition complex in the human fungal pathogen Candida albicans.

DNA replication in eukaryotes is initiated by the orchestrated assembly and association of initiator proteins (heterohexameric Origin Recognition Complex, ORC) on the replication origins. These functionally conserved proteins play significant roles in diverse cellular processes besides their central role in ignition of DNA replication at origins. Candida albicans, a major human fungal pathogen, is a diploid budding yeast that belongs to Ascomycota. However, C. albicans is significantly diverged from a well-studied model organism Saccharomyces cerevisiae, another ascomycete. The components of the DNA replication machinery in C. albicans remain largely uncharacterized. Identification of factors required for DNA replication is essential for understanding the evolution of the DNA replication machinery. We identified the putative ORC homologs in C. albicans and determined their relatedness with those of other eukaryotes including several yeast species. Our extensive in silico studies demonstrate that the domain architecture of CaORC proteins share similarities with the ORC proteins of S. cerevisiae. We dissect the domain organization of ORC (trans-acting factors) subunits that seem to associate with DNA replication origins in C. albicans. We present a model of the 3D structure of CaORC4 to gain further insights of this protein's function.

Facets of Autophagy Based Unconventional Protein Secretion-The Road Less Traveled.

Unconventional protein secretion (UCPS) of leaderless proteins bypasses the conventional endoplasmic reticulum (ER)-Golgi route. The proportion of UCPS in the secretome varies tremendously across eukaryotes. Interestingly, macroautophagy, an intracellular recycling process that is generally involved in cargo degradation, also participates in UCPS. This emerging field of secretory mode of autophagy is underexplored and has several unanswered questions regarding the composition of players, cargo, and the mechanisms that drive it. As secretomes vary considerably across cell types and physiological conditions, the contribution of secretory autophagy in healthy and pathophysiological states remain to be elucidated. Recent studies have begun to shed light on this enigmatic process.

Surface-Enhanced Raman Spectroscopy as a Tool for Distinguishing Extracellular Vesicles under Autophagic Conditions: A Marker for Disease Diagnostics.

Extracellular vesicles (EVs) laden with lipids, proteins, DNA, and micro-RNAs play important biological functions in intercellular communication and have pivotal roles in pathophysiological conditions. Characterization of the EVs has always been a multistep process involving large volumes, and they are heterogeneous in size and properties. A multitude of approaches is used to distinguish the EVs. Here, we report simple citrate reduced silver nanoparticles assisted surface-enhanced Raman spectroscopy (SERS) as a tool to distinguish EVs extracted from several cell lines isolated under autophagic conditions (nitrogen starvation). This study is the first report of its kind in characterizing EVs from cells under autophagic conditions using SERS. We used two cancerous cell lines, HeLa, its corresponding autophagy-deficient cell line (Atg5-/-), and a noncancerous cell line, HEK293, to isolate EVs. Our study helps in the facile detection and differentiation of EVs isolated between two closely related human cell lines that differ by their autophagic ability. The principal component analysis (PCA) of the SERS spectra of these EVs consistently showed the presence of distinct chemical compositions of the EVs. SERS of EVs can help in probing more into the molecular level information from EVs and could become a powerful tool once coupled with improved microscopy techniques for diagnosis and therapy.

Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins.

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid ('X' in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species--Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.

Analysis of stress-induced duplex destabilization (SIDD) properties of replication origins, genes and intergenes in the fission yeast, Schizosaccharomyces pombe.

BACKGROUND: Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe. RESULTS: We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD's role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region. CONCLUSION: SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.

Rapid evolution of Cse4p-rich centromeric DNA sequences in closely related pathogenic yeasts, Candida albicans and Candida dubliniensis.

The Cse4p-containing centromere regions of Candida albicans have unique and different DNA sequences on each of the eight chromosomes. In a closely related yeast, C. dubliniensis, we have identified the centromeric histone, CdCse4p, and shown that it is localized at the kinetochore. We have identified putative centromeric regions, orthologous to the C. albicans centromeres, in each of the eight C. dubliniensis chromosomes by bioinformatic analysis. Chromatin immunoprecipitation followed by PCR using a specific set of primers confirmed that these regions bind CdCse4p in vivo. As in C. albicans, the CdCse4p-associated core centromeric regions are 3-5 kb in length and show no sequence similarity to one another. Comparative sequence analysis suggests that the Cse4p-rich centromere DNA sequences in these two species have diverged faster than other orthologous intergenic regions and even faster than our best estimated "neutral" mutation rate. However, the location of the centromere and the relative position of Cse4p-rich centromeric chromatin in the orthologous regions with respect to adjacent ORFs are conserved in both species, suggesting that centromere identity is not solely determined by DNA sequence. Unlike known point and regional centromeres of other organisms, centromeres in C. albicans and C. dubliniensis have no common centromere-specific sequence motifs or repeats except some of the chromosome-specific pericentric repeats that are found to be similar in these two species. We propose that centromeres of these two Candida species are of an intermediate type between point and regional centromeres.