Biology subjects

Salemi, M. R.

Publications and source records attributed to Salemi, M. R..

3 recordsLinked to original sources

bioRxiv2021

Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy

The protein TRIM5 has multiple roles in anti-retroviral defense, but the mechanisms underlying TRIM5 action are unclear. Here, we used an APEX2-based proteomics approach to identify TRIM5-interacting proteins. Analysis of the TRIM5 interactome found proteins participating in a wide variety of cellular functions including regulating antiviral signaling pathways. We used this data set to uncover a novel role for TRIM5 in mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in multiple human diseases. Mitochondrial damage triggered the relocalization of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalized with markers of autophagy initiation and autophagosome biogenesis. Furthermore, we found that TRIM5 knockout attenuated both Parkin-dependent and Parkin-independent mitophagy by preventing the recruitment of autophagy regulators FIP200 and ATG13 to unhealthy mitochondria. Finally, TRIM5 knockout cells showed reduced mitochondrial function under basal conditions and were more susceptible to uncontrolled immune activation and cell death in response to mitochondrial damage than were wild type cells. Taken together, our studies have identified a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

cell biology↗

bioRxiv2021

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of matching non-synonymous single nucleotide polymorphisms for the individual who originated the protein sample. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of genetically variant peptides increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data independent acquisition and a variant of parallel reaction monitoring on a Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring on an Agilent 6495 triple quadrupole system. In our hands, average genetically variant peptide detection from a selected 24 genetically variant peptide panel increased from 6.5 {+/-} 1.1 and 3.1 {+/-} 0.8 using data dependent and independent acquisition to 9.5 {+/-} 0.7 and 11.7 {+/-} 1.7 using parallel reaction monitoring and multiple reaction monitoring (p < 0.05). Parallel reaction monitoring resulted in a 1.3-fold increase in detection sensitivity, and multiple reaction monitoring resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For parallel reaction monitoring and multiple reaction monitoring, detected genetically variant peptides were validated by the inclusion of stable isotope labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping, and the establishment of validation methods for the evaluation of inferred genotypes. HighlightsO_LITest four mass spectrometry configurations to optimize detection of genetically variant peptides C_LIO_LITechnology transfer of proteomic genotyping assays C_LIO_LIImproved sensitivity results in higher level of forensic discrimination for human identification using multiple reaction monitoring C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/435505v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1001cfaorg.highwire.dtl.DTLVardef@6e8aa1org.highwire.dtl.DTLVardef@14f8076org.highwire.dtl.DTLVardef@7ad930_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Proximity Biotin Labeling Reveals KSHV Interferon Regulatory Factor Networks

Studies on "HIT&RUN" effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposis sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses. ImportanceViral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects - those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

microbiology↗